Combinations of phosphoinositide 3-kinase inhibitor compounds chemotherapeutic agents, and methods of use

ABSTRACT

Combinations of PI3K inhibitor compounds having Formulas I and II and chemotherapeutic agents, including stereoisomers, geometric isomers, tautomers, metabolites and pharmaceutically acceptable salts thereof, are useful for treating hyperproliferative disorders such as cancer. Methods of using such combinations for in vitro, in situ, and in vivo diagnosis, prevention or treatment of such disorders in mammalian cells, or associated pathological conditions, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/208,227, filed 10Sep. 2008, which claims the benefit under 35 USC §119(e) of U.S.Provisional Application Ser. No. 60/971,773 filed on 12 Sep. 2007, eachof which are incorporated by reference in entirety.

FIELD OF THE INVENTION

The invention relates generally to pharmaceutical combinations ofcompounds with activity against hyperproliferative disorders such ascancer and which include compounds that inhibit PI3 kinase activity. Theinvention also relates to methods of using the compounds for in vitro,in situ, and in vivo diagnosis or treatment of mammalian cells, orassociated pathological conditions.

BACKGROUND OF THE INVENTION

Phosphatidylinositol is one of a number of phospholipids found in cellmembranes, and which participate in intracellular signal transduction.Cell signaling via 3′-phosphorylated phosphoinositides has beenimplicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity(Rameh et al (1999) J. Biol Chem. 274:8347-8350). The enzyme responsiblefor generating these phosphorylated signaling products,phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K),was originally identified as an activity associated with viraloncoproteins and growth factor receptor tyrosine kinases thatphosphorylate phosphatidylinositol (PI) and its phosphorylatedderivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al(1992) Trends Cell Biol 2:358-60). Phosphoinositide 3-kinases (PI3K) arelipid kinases that phosphorylate lipids at the 3-hydroxyl residue of aninositol ring (Whitman et al (1988) Nature, 332:664). The3-phosphorylated phospholipids (PIP3s) generated by PI3-kinases act assecond messengers recruiting kinases with lipid binding domains(including plekstrin homology (PH) regions), such as Akt and PDK1,phosphoinositide-dependent kinase-1 (Vivanco et al (2002) Nature Rev.Cancer 2:489; Phillips et al (1998) Cancer 83:41).

The PI3 kinase family comprises at least 15 different enzymessub-classified by structural homology and are divided into 3 classesbased on sequence homology and the product formed by enzyme catalysis.The class I PI3 kinases are composed of 2 subunits: a 110 kd catalyticsubunit and an 85 kd regulatory subunit. The regulatory subunits containSH2 domains and bind to tyrosine residues phosphorylated by growthfactor receptors with a tyrosine kinase activity or oncogene products,thereby inducing the PI3K activity of the p110 catalytic subunit whichphosphorylates its lipid substrate. Class I PI3 kinases are involved inimportant signal transduction events downstream of cytokines, integrins,growth factors and immunoreceptors, which suggests that control of thispathway may lead to important therapeutic effects such as modulatingcell proliferation and carcinogenesis. Class I PI3Ks can phosphorylatephosphatidylinositol (PI), phosphatidylinositol-4-phosphate, andphosphatidylinositol-4,5-biphosphate (PIP2) to producephosphatidylinositol-3-phosphate (PIP),phosphatidylinositol-3,4-biphosphate, andphosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ksphosphorylate PI and phosphatidylinositol-4-phosphate. Class III PI3Kscan only phosphorylate PI. The main PI3-kinase isoform in cancer is theClass I PI3-kinase, p110α. (U.S. Pat. No. 5,824,492; U.S. Pat. No.5,846,824; U.S. Pat. No. 6,274,327). Other isoforms are implicated incardiovascular and immune-inflammatory disease (Workman P (2004) BiochemSoc Trans 32:393-396; Patel et al (2004) Proc. Am. Assoc. of Cancer Res.(Abstract LB-247) 95th Annual Meeting, March 27-31, Orlando, Fla., USA;Ahmadi K and Waterfield M D (2004) “Phosphoinositide 3-Kinase: Functionand Mechanisms” Encyclopedia of Biological Chemistry (Lennarz W J, LaneM D eds) Elsevier/Academic Press), and oncogene mutations in PI3 kinase(Samuels et al (2004) Science 304:554). Oncogenic mutations of p110alpha have been found at a significant frequency in colon, breast,brain, liver, ovarian, gastric, lung, and head and neck solid tumors.PTEN abnormalities are found in glioblastoma, melanoma, prostate,endometrial, ovarian, breast, lung, head and neck, hepatocellular, andthyroid cancers.

The initial purification and molecular cloning of PI3 kinase revealedthat it was a heterodimer consisting of p85 and p110 subunits (Otsu etal (1991) Cell 65:91-104; Hiles et al (1992) Cell 70:419-29). Sincethen, four distinct Class I PI3Ks have been identified, designated PI3Kα (alpha), β (beta), δ (delta), and ω (gamma), each consisting of adistinct 110 kDa catalytic subunit and a regulatory subunit. Morespecifically, three of the catalytic subunits, i.e., p110 alpha, p110beta and p110 delta, each interact with the same regulatory subunit,p85; whereas p110 gamma interacts with a distinct regulatory subunit,p101. The patterns of expression of each of these PI3Ks in human cellsand tissues are also distinct. In each of the PI3K alpha, beta, anddelta subtypes, the p85 subunit acts to localize PI3 kinase to theplasma membrane by the interaction of its SH2 domain with phosphorylatedtyrosine residues (present in an appropriate sequence context) in targetproteins (Rameh et al (1995) Cell, 83:821-30; Volinia et al (1992)Oncogene, 7:789-93).

The PI3 kinase/Akt/PTEN pathway is an attractive target for cancer drugdevelopment since such agents would be expected to inhibitproliferation, reverse the repression of apoptosis and surmountresistance to cytotoxic agents in cancer cells. PI3 kinase inhibitorshave been reported (Yaguchi et al (2006) Jour. of the Nat. Cancer Inst.98(8):545-556; U.S. Pat. Nos. 7,173,029; 7,037,915; 6,608,056;6,608,053; 6,838,457 ; 6,770,641; 6,653,320; 6,403,588; WO 2006/046031;WO 2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP08176070). Wortmannin analogs have PI3 kinase activity in mammals (U.S.Pat. No. 6,703,414; WO 97/15658).

Thienopyrimidine compounds of Formulas I and II have p110 alpha binding,PI3 kinase inhibitory activity and inhibit the growth of cancer cells(WO 2006/046031; US 2008/0039459; US 2008/0076768; US 2008/0076758; WO2008/070740; WO 2008/073785.

Formula I compound, GDC-0941 (Genentech Inc.), is a selective, orallybioavailable inhibitor of PI3K with promising pharmacokinetic andpharmaceutical properties (Belvin et al, American Association for CancerResearch Annual Meeting 2008, 99th: April 15, Abstract 4004; Folkes etal, American Association for Cancer Research Annual Meeting 2008, 99th:April 14, Abstract LB-146; Friedman et al, American Association forCancer Research Annual Meeting 2008, 99th: April 14, Abstract LB-110).

Combinations of anti-cancer pharmaceutical therapeutics administeredsimultaneously or sequentially in a dosing regimen are now common incancer treatment. Successful combination therapy provides improved andeven synergistic effect over mono-therapy, i.e. pharmaceutical treatmentlimited to one drug. Combination therapy for the treatment ofhyperproliferative disorders such as cancer has been studied, includingantitumor activity of erlotinib in combination with capecitabine inhuman tumor xenograft models (Ouchi et al (2006) Cancer Chemother.Pharmacol. 57:693-702), and erlotinib in combination gemcitabine andcisplatin in non-small cell lung cancer (NSCLC) tumor xenograft models(Higgins et al (2004) Anti-Cancer Drugs 15:503-512). Preclinicalresearch has been the basis for prediction of clinical stage synergy ofanti-cancer pharmaceutical therapeutic combinations includingcapecitabine and taxanes for the treatment of breast cancer (Sawada etal (1998) Clin. Cancer Res. 4:1013-1019). Certain doses and schedules ofcombination therapy of capecitabine and taxane can improve safetywithout compromising efficacy (O'Shaughnessy et al (2006) Clin. BreastCancer April 7(1):42-50). Synergistic effects of anti-fungalcombinations in vitro have been correlated with clinical stage synergy(Steinbach et al (2003) Clin. Inf. Dis. October 1; 37 Suppl 3:S188-224).

SUMMARY OF THE INVENTION

The invention relates generally to thienopyrimidine compounds ofFormulas I and II with anti-cancer activity, and more specifically withPI3 kinase inhibitory activity, administered in combination withchemotherapeutic agents to inhibit the growth of cancer cells. Certaincombinations of Formula I and II compounds with chemotherapeutic agentsshow synergistic effects in inhibiting the growth of cancer cells invitro and in vivo. The combinations and methods of the invention may beuseful in the treatment of hyperproliferative disorders such as cancer.The compositions may inhibit tumor growth in mammals and may be usefulfor treating human cancer patients.

In one aspect, the invention includes a method for the treatment of ahyperproliferative disorder comprising administering a therapeuticcombination as a combined formulation or alternation to a mammal,wherein the therapeutic combination comprises a therapeuticallyeffective amount of a compound having Formula I or II, and atherapeutically effective amount of a chemotherapeutic agent selectedfrom erlotinib, docetaxel, 5-FU, gemcitabine, PD-0325901, cisplatin,carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab,temozolomide, tamoxifen, doxorubicin, Akti-1/2, HPPD, rapamycin, andlapatinib.

The invention also relates to methods of using the compositions for invitro, in situ, and in vivo diagnosis or treatment of mammalian cells,organisms, or associated pathological conditions.

An aspect of the invention provides therapeutic combinations comprising4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine(US 2008/0076768; WO 2006/046031), also known as GDC-0941 (Genentech,Inc.) and having Formula Ia and a therapeutically effective amount of achemotherapeutic agent selected from erlotinib, docetaxel, 5-FU,gemcitabine, PD-0325901, cisplatin, carboplatin, paclitaxel,bevacizumab, trastuzumab, pertuzumab, temozolomide, tamoxifen,doxorubicin, Akti-1/2, HPPD, rapamycin, and lapatinib.

An aspect of the invention provides therapeutic combinations comprising(S)-1-(4-42-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one(WO 2008/070740) having Formula Ib and a therapeutically effectiveamount of a chemotherapeutic agent selected from erlotinib, docetaxel,5-FU, gemcitabine, PD-0325901, cisplatin, carboplatin, paclitaxel,bevacizumab, trastuzumab, pertuzumab, temozolomide, tamoxifen,doxorubicin, Akti-1/2, HPPD, rapamycin, and lapatinib.

An aspect of the invention provides therapeutic combinations comprising4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine(US 2008/0076758; WO 2006/046031) having Formula IIa and atherapeutically effective amount of a chemotherapeutic agent selectedfrom erlotinib, docetaxel, 5-FU, gemcitabine, PD-0325901, cisplatin,carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab,temozolomide, tamoxifen, doxorubicin, Akti-1/2, HPPD, rapamycin, andlapatinib.

Formula Ia, Ib, and IIa compounds are orally bioavailable and havesingle agent anti-tumor activity in multiple human cancer models.

Formula I and II compounds include all stereoisomers, geometric isomers,tautomers, metabolites, and pharmaceutically acceptable salts thereof.Certain Formula I and II compounds are potent inhibitors of PI3K withdrug-like physicochemical and pharmacokinetic properties. CertainFormula I and II compounds exhibit selectivity for class Ia PI3Ks overclass Ib, in particular for the P110 alpha subtype.

Pharmaceutical compositions and therapeutic combinations of theinvention comprise a chemotherapeutic agent selected from erlotinib,docetaxel, 5-FU, gemcitabine, PD-0325901, cisplatin, carboplatin,paclitaxel, bevacizumab, trastuzumab, pertuzumab, temozolomide,tamoxifen, doxorubicin, Akti-1/2, HPPD, rapamycin, and lapatinib.

Pharmaceutical compositions of the invention may further comprise apharmaceutically acceptable carrier.

Another aspect of the invention provides methods of treating ahyperproliferative disease or disorder modulated by PI3 kinases,comprising administering to a mammal in need of such treatment effectiveamounts of a Formula I or II compound and a chemotherapeutic agent. TheFormula I or II compound and the chemotherapeutic agent may beco-formulated for administration in a combination as a pharmaceuticalcomposition or they may be administered separately in alternation(sequentially) as a therapeutic combination.

Another aspect of the invention provides methods of treating ahyperproliferative disorder, comprising administering to a mammal inneed of such treatment effective amounts of the Formula I or II compoundand a chemotherapeutic agent.

In a further aspect the present invention provides a method of using apharmaceutical composition of the invention to treat a disease orcondition modulated by PI3 kinase in a mammal.

An additional aspect of the invention is the use of a pharmaceuticalcomposition of the invention in the preparation of a medicament for thetreatment of a disease or condition modulated by PI3 kinase in a mammal.

Another aspect of the invention includes articles of manufacture or kitscomprising a Formula I or II compound, a chemotherapeutic agent, acontainer, and optionally a package insert or label indicating atreatment.

Another aspect of the invention is a product comprising a compoundhaving Formula I or II, and a chemotherapeutic agent selected fromerlotinib, docetaxel, 5-FU, gemcitabine, PD-0325901, cisplatin,carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab,temozolomide, tamoxifen, doxorubicin, Akti-1/2, HPPD, rapamycin, andlapatinib; as a combined preparation for separate, simultaneous orsequential use in the treatment of a hyperproliferative disorder.

Another aspect of the invention includes a method for determiningcompounds to be used in combination for the treatment of cancercomprising: a) administering a therapeutic combination of a compoundhaving Formula I or II, and a chemotherapeutic agent to HER2-amplifiedbreast cancer cells in laminin-rich, reconstituted basement membranemedia, wherein the chemotherapeutic agent targets, binds to, ormodulates a HER2 receptor, and b) measuring inhibition of cellularproliferation wherein nonmalignant and malignant mammary cells arediscriminated by one or more phenotypic difference selected from cellviability and acinar morphogenesis.

Another aspect of the invention includes a method for determiningcompounds to be used in combination for the treatment of cancercomprising: a) administering a therapeutic combination of claim 1 to anin vitro tumor cell line with a K-ras mutation, and b) measuring asynergistic or non-synergistic effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-A shows a summary of in vitro cell proliferation assays ofcombinations of Formula Ia compound and various chemotherapeutic agents,dosed simultaneously. The cell lines are characterized by tumor type andpresence of known mutation. The individual measured EC50 values of thechemotherapeutic agent and the Formula Ia compound (GDC-0941) arecompared to the combination EC50 value and a combination index score iscalculated by the Chou and Talalay method (Chou, T. and Talalay, P.(1984) Adv. Enzyme Regul. 22:27-55). The strength of synergy is scoredusing the ranking system and is listed in the last column.

FIG. 1-B shows a summary of in vitro cell proliferation assays ofcombinations of Formula IIa compound and various chemotherapeuticagents. The cell lines are characterized by tumor type and presence ofRas mutation. The individual measured EC50 values of thechemotherapeutic agent and the Formula IIa compound are compared to thecombination EC50 value and a combination index score is calculated bythe Chou and Talalay method (Chou, T. and Talalay, P. (1984) Adv. EnzymeRegul. 22:27-55). The strength of synergy is scored using the rankingsystem from Chou and Talalay.

FIG. 1-C shows a summary of in vitro cell proliferation assays ofcombinations of Formula Ib compound and various chemotherapeutic agents.The cell lines are characterized by tumor type and presence of Rasmutation. The individual measured EC50 values of the chemotherapeuticagent and the Formula Ib compound are compared to the combination EC50value and a combination index score is calculated by the Chou andTalalay method (Chou, T. and Talalay, P. (1984) Adv. Enzyme Regul.22:27-55). The strength of synergy is scored using the ranking systemfrom Chou and Talalay.

FIG. 2 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of 5-FU, Formula Ia compound(GDC-0941), and the combination of 5-FU and Formula Ia. The MDA-MB-361(breast tumor type) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with 5-FU (middle), and post-dosedwith Formula Ia 4 hours after dosing with 5-FU (bottom).

FIG. 3 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of gemcitabine, Formula Ia compound(GDC-0941), and the combination of gemcitabine and Formula Ia. TheCal-51 (breast tumor type) cells are treated simultaneously (top), andpost-dosed with Formula Ia 4 hours after dosing with gemcitabine(bottom).

FIG. 4 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of gemcitabine, Formula Ia compound(GDC-0941), and the combination of gemcitabine and Formula Ia. TheMDA-MB-361 (breast tumor type) cells are treated simultaneously (top),pre-dosed with Formula Ia 4 hours before dosing with gemcitabine(middle), and post-dosed with Formula Ia 4 hours after dosing withgemcitabine (bottom).

FIG. 5 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of erlotinib, Formula Ia compound(GDC-0941), and the combination of erlotinib and Formula Ia. The A549(lung tumor type with K-ras G12C) cells are treated simultaneously(top), pre-dosed with Formula Ia 4 hours before dosing with erlotinib(middle), and post-dosed with Formula Ia 4 hours after dosing witherlotinib (bottom).

FIG. 6 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of erlotinib, Formula Ia compound(GDC-0941), and the combination of erlotinib and Formula Ia. The H23(lung tumor type, with K-ras G12C mutation) cells are treatedsimultaneously (top), pre-dosed with Formula Ia 4 hours before dosingwith erlotinib (middle), and post-dosed with Formula Ia 4 hours afterdosing with erlotinib (bottom).

FIG. 7 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of temozolomide, Formula Ia compound(GDC-0941), and the combination of temozolomide and Formula Ia. The U87(glioma tumor type) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with temozolomide (middle), andpost-dosed with Formula Ia 4 hours after dosing with temozolomide(bottom).

FIG. 8 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of temozolomide, Formula Ia compound(GDC-0941), and the combination of temozolomide and Formula Ia. The A375(melanoma tumor type) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with temozolomide (middle), andpost-dosed with Formula Ia 4 hours after dosing with temozolomide(bottom).

FIG. 9 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of temozolomide, Formula Ia compound(GDC-0941), and the combination of temozolomide and Formula Ia. TheMALME-3M (melanoma tumor type) cells are treated simultaneously (top),pre-dosed with Formula Ia 4 hours before dosing with temozolomide(middle), and post-dosed with Formula Ia 4 hours after dosing withtemozolomide (bottom).

FIG. 10 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of doxorubicin, Formula Ia compound(GDC-0941), and the combination of doxorubicin and Formula Ia. The SKOV3(ovarian tumor type) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with doxorubicin (middle), andpost-dosed with Formula Ia 4 hours after dosing with doxorubicin(bottom).

FIG. 11 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of docetaxel, Formula Ia compound(GDC-0941), and the combination of docetaxel and Formula Ia. The PC3(prostate tumor type) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with docetaxel (middle), andpost-dosed with Formula Ia 4 hours after dosing with docetaxel (bottom).

FIG. 12 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable MDA-MB 361 (breast tumor type) cells overvarying concentrations (starting at 4× EC50) right to left of: (top)5-FU, Formula IIa compound, and the simultaneous combination of 5-FU andFormula IIa); (middle) docetaxel, Formula IIa compound, and thesimultaneous combination of docetaxel and Formula IIa; and (bottom)gemcitabine, Formula IIa compound, and the simultaneous combination ofgemcitabine and Formula IIa.

FIG. 13 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring: (top) viable MT3 (breast tumor type) cells overvarying concentrations (starting at 4× EC50) right to left of docetaxel,Formula IIa compound, and the simultaneous combination of docetaxel andFormula IIa; and (bottom) viable U87 (glioma tumor type) cells overvarying concentrations (starting at 4× EC50) right to left oftemozolomide, Formula IIa compound, and the simultaneous combination oftemozolomide and Formula IIa.

FIG. 14 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable ZR75-1 (breast tumor type) cells overvarying concentrations (starting at 4× EC50) right to left of: (top)5-FU, Formula IIa compound, and the simultaneous combination of 5-FU andFormula IIa; and (bottom) docetaxel, Formula IIa compound, and thesimultaneous combination of docetaxel and Formula IIa.

FIG. 15 shows a dot plot of synergy (Combination Index) of erlotinib andFormula Ia compound (GDC-0941) experiments from FIG. 1-A against tumorcell lines without Ras mutations (Ras WT, Expts. 41, 42, 73-75, 77,79-81, 83, 84, 86) and with Ras mutations (Ras Mut, Expts. 40, 69-72,76, 78, 82, 144, 145).

FIG. 16 shows a dot plot of synergy (Combination Index) of PD-0325901and Formula Ia compound (GDC-0941) experiments from FIG. 1-A againsttumor cell lines without Ras mutations (Ras WT, Expts. 22-24, 26-28,31-33, 36-38, 55, 59, 61, 63-66, 85, 89-98, 149, 161, 162) and with Rasmutations (Ras Mut, Expts. 25, 30, 34, 35, 39, 56-58, 60, 62, 67, 68,146-148, 150).

FIG. 17 shows time-course results of treatment of a synergistic tumorcell line MDA-MB-361 and a non-synergistic tumor cell line MT-3 withgemcitabine at EC80 dosing levels. pAKT levels were measured at T=0(untreated, UT), 1 hr, 4 hr, 6 hr, and 24 hr.

FIG. 18 shows a dot plot of synergy (Combination Index) of docetaxel,5-FU, or gemcitabine and Formula Ia compound (GDC-0941) experiments fromFIG. 1-A against tumor cell lines that show a pAkt increase or no pAktincrease.

FIG. 19 shows results of flow cytometry FACS (Fluorescence ActivatedCell Sorter): (top) MB361 cells are (left to right) untreated, treatedwith Formula Ia, 5FU, and first with 5FU then Formula Ia compound(GDC-0941); (middle) PC3 cells are (left to right) untreated, treatedwith Formula Ia, docetaxel, simultaneous with Formula Ia compound anddocetaxel, first with Formula Ia then with docetaxel, and first withdocetaxel, then with Formula Ia; and (bottom) MB361 cells are (left toright) untreated, treated Formula Ia, gemcitabine, and first withgemcitabine then with Formula Ia.

FIG. 20 shows treatment of BT474M1 cells in three dimensional (3D)culture. Acini growth and morphogenesis is correlated with cellular ATPproduction in relative light units (RLU) in 10% FBS medium with andwithout 1 nM heregulinl by treatment with (left to right): media, DMSO,combination of 20 μg/ml trastuzumab and 25 μg/ml pertuzumab, 250 nMFormula Ia compound (GDC-0941), and the combination of 20 μg/mltrastuzumab, 25 μg/ml pertuzumab, and 250 nM Formula Ia compound.

FIG. 21 shows treatment of BT474M1 cells in three dimensional (3D)culture. Acini growth and morphogenesis is correlated with cellular ATPproduction in relative light units (RLU) in 10% FBS medium with andwithout 1 nM heregulinl by treatment with (left to right): media, DMSO,combination of 20 μg/ml trastuzumab and 25 μg/ml pertuzumab, 250 nMFormula IIa compound, and the combination of 20 μg/ml trastuzumab, 25μg/ml pertuzumab, and 250 nM Formula IIa compound.

FIG. 21-A shows treatment of BT474M1 cells in three dimensional (3D)culture. Acini growth and morphogenesis is correlated with cellular ATPproduction in relative light units (RLU) in 10% FBS medium with andwithout 1 nM heregulinl by treatment with (left to right): DMSO,combination of 20 gg/m1trastuzumab and 25 μg/ml pertuzumab, 20 nMFormula Ib compound, and the combination of 20 Wall trastuzumab, 25μg/ml pertuzumab, and 20 nM Formula Ib compound.

FIG. 22 shows the mean tumor volume change over time in CD-1 nude mice(Charles River Labs) with MDA-MB-361.1 breast tumor cell xenograftsdosed on day 0 with: MCT Vehicle (0.5% methylcellulose/0.2% Tween 80),150 mg/kg Formula Ia (GDC-0941), 5 mg/kg docetaxel, and the combinationof Formula Ia 150 mg/kg and docetaxel 5 mg/kg. Mice were dosed withdocetaxel on day 1, 5 and 9 (q4d×3) intravenously while Formula Ia wasdosed daily for 21 days by oral gavage.

FIG. 23 shows the mean tumor volume change over time in CD-1 nude mice(Charles River Labs) with MDA-MB-361.1 breast tumor cell xenograftsdosed on day 1 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80),37.5 mg/kg Formula IIa , 5 mg/kg docetaxel and the combination of 37.5mg/kg Formula IIa and 5 mg/kg. Mice were dosed with docetaxel on day 1,5 and 9 (q4d×3) intravenously while Formula IIa was dosed daily for 21days by oral gavage.

FIG. 24 shows the mean tumor volume change over time in NMRI femalenu/nu (nude) mice with MAXF 401 primary breast tumor explant xenograftsdosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80),100 mg/kg Formula Ia (GDC-0941), 15 mg/kg docetaxel and the combinationof 100 mg/kg Formula Ia and 15 mg/kg docetaxel. Mice were dosedintravenously with docetaxel on day 0 and day 11 while Formula Ia wasdosed on day 0-4, 11-17 and 21-28 by oral gavage.

FIG. 25 shows the mean tumor volume change over time in NMRI femalenu/nu nude mice with MAXF 401 primary breast tumor explant xenograftsdosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80),100 mg/kg Formula IIa , 15 mg/kg docetaxel and the combination of 100mg/kg Formula IIa and 15 mg/kg docetaxel. Mice were dosed intravenouslywith docetaxel on day 0 and day 11 while Formula IIa was dosed on day0-3, 11-17 and 21-28 by oral gavage.

FIG. 26 shows the mean tumor volume change over time in NMRI femalenu/nu nude mice with MAXF 1162 primary breast tumor explant xenograftsdosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80),100 mg/kg Formula Ia (GDC-0941), 15 mg/kg docetaxel and the combinationof 100 mg/kg Formula Ia and 15 mg/kg docetaxel. Mice were dosedintravenously with docetaxel on day 0, 11, 22 and 44 and Formula Ia wasdosed on day 0-5, 11-16, 22-27, 30-32, 42 and 44 by oral gavage.

FIG. 27 shows the mean tumor volume change over time in NMRI femalenu/nu nude mice with MAXF 1162 primary breast tumor xenografts dosed onday 0 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80), 100 mg/kgFormula IIa, 15 mg/kg docetaxel and the combination of 100 mg/kg FormulaIIa and 15 mg/kg docetaxel. Mice were dosed intravenously with docetaxelon day 0, 11, 22 and 44 and Formula IIa was dosed on day 0-5, 11-16,22-23, 29-31 and 35-38 by oral gavage.

FIG. 28 shows the mean tumor volume change over time in CRL female nu/nu(nude) mice with NCI-H2122 non-small cell lung cancer (NSCLC) tumorxenografts dosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2%Tween 80), 50 mg/kg Formula Ia (GDC-0941), 75 mg/kg erlotinib and thecombination of 50 mg/kg Formula Ia and 75 mg/kg erlotinib. Mice weredosed daily with erlotinib and Formula Ia daily for 16 days by oralgavage.

FIG. 29 shows the mean tumor volume change over time in CRL female nu/nu(nude) mice with NCI-H2122 non-small cell lung cancer (NSCLC) tumorxenografts dosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2%Tween 80), 50 mg/kg Formula IIa, 75 mg/kg erlotinib and the combinationof 50 mg/kg Formula IIa and 75 mg/kg erlotinib. Mice were dosed dailywith erlotinib and Formula IIa for 14 days (end of study) by oralgavage.

FIG. 30 shows the mean tumor volume change over time with HRLN femalenu/nu mice with MCF-7 (PI3K mutant) breast tumor cell xenografts dosedon day 0 with: MCT and PBS vehicle (MCT; 0.5% methycellulose/0.2% Tween80 and PBS; phosphate buffered saline), control IgG 5 mg/kg, mB20-4.1murine anti-VEGF 5 mg/kg, Formula Ia (GDC-0941) 150 mg/kg, and thecombination of Formula Ia 150 mg/kg and mB20-4.1 murine anti-VEGF 5mg/kg. Animals were dosed with control IgG and mB20-4.1 intraperitonealytwice a week for 3 weeks and with Formula Ia daily for 21 days by oralgavage and tumor growth was monitored for an additional 41 days (totalnumber of day on-study was 62). Formula Ia and mB20-4.1 wereco-administered simultaneously.

FIG. 31 shows the mean tumor volume change over time with HRLN femalenu/nu mice with MCF-7 (PI3K mutant) breast tumor cell xenografts dosedon day 0 with: MCT and PBS vehicle (MCT; 0.5% methycellulose/0.2% Tween80 and PBS; phosphate buffered saline), control IgG 5 mg/kg, mB20-4.1murine anti-VEGF (anti-angiogenic) 5 mg/kg, Formula IIa 100 mg/kg, andthe combination of Formula IIa 100 mg/kg and mB20-4.1 murine anti-VEGF 5mg/kg. Animals were dosed with control IgG and mB20-4.1 intraperitonealytwice a week for 3 weeks and with Formula IIa orally daily for 21 daysand tumor growth was monitored for an additional 41 days (total numberof day on-study was 62). Formula IIa and mB20-4.1 were co-administeredsimultaneously.

FIG. 32 shows the mean tumor volume change over time with Harlan femalenu/nu with U87MG glioma tumor cell xenografts dosed on day 0 with:Formula Ia (GDC-0941) 109 mg/kg, temozolomide 100 mg/kg, and thecombination of Formula Ia 109 mg/kg and temozolomide 100 mg/kg, alongwith mice receiving no drug (No Treatment group). Animals were dosedwith Formula Ia orally daily for 21 days, and temozolomide orally dailyfor 5 days.

FIG. 33 shows the mean tumor volume change over time with CD-1 nude micewith MDA-MB-361.1 breast tumor cell xenografts dosed on day 0 with:Formula Ia (GDC-0941) 150 mg/kg, gemcitabine 100 mg/kg, and thecombination of Formula Ia 150 mg/kg and gemcitabine 100 mg/kg, alongwith mice receiving no drug (Vehicle group). Animals were dosed withFormula Ia orally daily for 21 days, and gemcitabine intraperitoneal ondays 1, 4, 7 and 10 (q3d×4).

FIG. 34 shows the mean tumor volume change over time with Harlan femalenu/nu mice with BT474 breast tumor cell xenografts dosed on day 0 with:Formula Ia (GDC-0941) at 18, 36, and 73 mg/kg, trastuzumab 20 mg/kg, andthe combinations of Formula Ia at 18, 36, and 73 mg/kg and trastuzumab20 mg/kg, along with mice receiving no drug (Vehicle group). Animalswere dosed with Formula Ia orally daily for 21 days, and trastuzumabintravenously twice a week for 3 weeks.

FIG. 35 shows the mean tumor volume change over time with Harlan femalenu/nu mice with BT474 breast tumor cell xenografts dosed on day 0 with:Formula Ib at 2.5 mg/kg orally daily for 3 weeks, Formula Ib at 2.5mg/kg orally twice a week for 3 weeks, Formula Ib at 5 mg/kg orallydaily for 3 weeks, trastuzumab 15 mg/kg intravenously once a week for 3weeks, and the combinations of Formula Ib at 2.5 mg/kg orally daily for3 weeks and trastuzumab 15 mg/kg intravenously once a week for 3 weeks;Formula Ib at 2.5 mg/kg orally twice a week for 3 weeks and trastuzumab15 mg/kg intravenously once a week for 3 weeks; and Formula Ib at 5mg/kg orally daily for 3 weeks and trastuzumab 15 mg/kg intravenouslyonce a week for 3 weeks, along with mice receiving no drug (Vehiclegroup).

FIG. 36 shows the mean tumor volume change over time with Harlan femalenu/nu mice with MCF-7 breast tumor cell xenografts dosed on day 0 with:murine anti-VEGF antibody B20-4.1 5 mg/kg intraperitoneal twice a weekfor 3 weeks, Formula Ib at 10 mg/kg orally daily for 4 days, Formula Ibat 5 mg/kg orally daily for days 0-3, 10-26, Formula Ib at 2 mg/kgorally daily for days 0-4, 10-25, and the combinations of: Formula Ib at10 mg/kg orally daily for 4 days and B20-4.1 5 mg/kg intraperitonealtwice a week for 3 weeks; Formula Ib at 5 mg/kg orally daily for days0-3, 10-26 and B20-4.1 5 mg/kg intraperitoneal twice a week for 3 weeks;and Formula Ib at 2 mg/kg orally daily for days 0-4, 10-25 and B20-4.1 5mg/kg intraperitoneal twice a week for 3 weeks, along with micereceiving no drug (Vehicle group).

FIG. 37 shows the mean tumor volume change over time with Harlan femalenu/nu mice with Fo5 breast tumor cell xenografts dosed on day 0 with:murine anti-VEGF antibody B20-4.1 5 mg/kg intraperitoneal twice a weekfor 3 weeks, Formula Ia (GDC-0941) at 36 and 73 mg/kg orally daily for21 days, Formula Ib at 2.5 and 5 mg/kg orally daily for 21 days, and thecombinations of: Formula Ia at 36 mg/kg orally daily for 21 days andB20-4.1 5 mg/kg intraperitoneal twice a week for 3 weeks; Formula Ia at73 mg/kg orally daily for 21 days and B20-4.1 5 mg/kg intraperitonealtwice a week for 3 weeks; Formula Ib at 5 mg/kg orally daily for 21 daysand B20-4.1 5 mg/kg intraperitoneal twice a week for 3 weeks, andFormula Ib at 2.5 mg/kg orally daily for 21 days and B20-4.1 5 mg/kgintraperitoneal twice a week for 3 weeks along with mice receiving nodrug (Vehicle group).

FIG. 38 shows the mean tumor volume change over time with Harlan femalenu/nu mice with MDA-MB-231 breast tumor cell xenografts dosed on day 0with: murine anti-VEGF antibody B20-4.1 5 mg/kg intraperitoneal twice aweek for 3 weeks, Formula Ia (GDC-0941) at 36 and 73 mg/kg orally dailyfor 21 days, Formula Ib at 5 mg/kg orally daily for 21 days and FormulaIb at 7.5 mg/kg orally daily for 8 days, and the combinations of:Formula Ia at 36 mg/kg orally daily for 21 days and B20-4.1 5 mg/kgintraperitoneal twice a week for 3 weeks; Formula Ia at 73 mg/kg orallydaily for 21 days and B20-4.1 5 mg/kg intraperitoneal twice a week for 3weeks; Formula Ib at 5 mg/kg orally daily for 21 days and B20-4.1 5mg/kg intraperitoneal twice a week for 3 weeks; and Formula Ib at 7.5mg/kg orally daily for 8 days and B20-4.1 5 mg/kg intraperitoneal twicea week for 1.5 weeks, along with mice receiving no drug (Vehicle group).

FIG. 39 shows the mean tumor volume change over time with Harlan femalenu/nu mice with H1299 non-small cell lung cancer (NSCLC) tumor cellxenografts dosed on day 0 with: erlotinib 50 mg/kg orally daily for 21days, Formula Ia (GDC-0941) at 100 mg/kg orally daily for 6 days,Formula Ia 50 mg/kg orally daily for 21 days, Formula Ia 25 mg/kg orallydaily for 21 days, and the combinations of Formula Ia 100 mg/kg orallydaily for 6 days and erlotinib 50 mg/kg orally daily for 6 days; FormulaIa 50 mg/kg orally daily for 21 days and erlotinib 50 mg/kg orally dailyfor 21 days; and Formula Ia 25 mg/kg orally daily for 21 days anderlotinib 50 mg/kg orally daily for 21 days, along with mice receivingno drug (Vehicle group).

FIG. 40 shows the mean tumor volume change over time with Harlan femalenu/nu mice with H520 non-small cell lung cancer (NSCLC) tumor cellxenografts dosed on day 0 with: erlotinib 50 mg/kg orally daily for 21days, Formula Ia (GDC-0941) at 73 mg/kg orally daily for 4 days, FormulaIa at 36 mg/kg orally daily for 21 days, Formula Ia 18 mg/kg orallydaily for 21 days, and the combinations of Formula Ia 73 mg/kg orallydaily for 4 days and erlotinib 50 mg/kg orally daily for 4 days; FormulaIa 36 mg/kg orally daily for 21 days and erlotinib 50 mg/kg orally dailyfor 21 days; and Formula Ia 18 mg/kg orally daily for 21 days anderlotinib 50 mg/kg orally daily for 21 days, along with mice receivingno drug (Vehicle group).

FIG. 41 shows the mean tumor volume change over time with Harlan femalenu/nu mice with H1299 non-small cell lung cancer (NSCLC) tumor cellxenografts dosed on day 0 with: erlotinib 50 mg/kg orally daily for 3weeks, Formula Ib at 2.5 mg/kg orally daily for 21 days, Formula Ib at 5mg/kg orally twice per week for 3 weeks, Formula Ib at 5 mg/kg orallyonce per week for 3 weeks, and the combinations of: Formula Ib at 2.5mg/kg orally daily for 21 days and erlotinib 50 mg/kg orally daily for 3weeks; Formula Ib at 5 mg/kg orally twice per week for 3 weeks anderlotinib 50 mg/kg orally daily for 3 weeks; and Formula Ib at 5 mg/kgorally once per week for 3 weeks and erlotinib 50 mg/kg orally daily for3 weeks, along with mice receiving no drug (Vehicle group).

FIG. 42 shows the mean tumor volume change over time with Taconic NCRfemale nude mice with NCI-H2122 non-small cell lung cancer (NSCLC) tumorcell xenografts dosed on day 0 with: erlotinib 75 mg/kg orally daily for16 days, Formula Ib at 2.5 mg/kg orally daily for 16 days, Formula Ib at5 mg/kg orally daily for 16 days, Formula Ib at 7.5 mg/kg orally dailyfor 16 days, and the combinations of: Formula Ib at 2.5 mg/kg orallydaily for 16 days and erlotinib 50 mg/kg orally daily for 3 weeks;Formula Ib at 5 mg/kg orally twice per week for 3 weeks and erlotinib 50mg/kg orally daily for 16 days; and Formula Ib at 5 mg/kg orally dailyfor 16 days and erlotinib 50 mg/kg orally daily for 16 days, along withmice receiving no drug (Vehicle group).

FIG. 43 shows the mean tumor volume change over time with Harlan femalenu/nu mice with A375 human melanoma cancer tumor cell xenografts dosedon day 0 with: PD-0325901 3 mg/kg orally daily for 3 weeks, Formula Ia(GDC-0941) at 73 mg/kg orally daily for 3 weeks, and the combination of:PD-0325901 3 mg/kg orally daily for 3 weeks and Formula Ia 73 mg/kgorally daily for 3 weeks, along with mice receiving no drug (Vehiclegroup).

FIG. 44 shows the mean tumor volume change over time with Harlan femalenu/nu mice with A375 human melanoma cancer tumor cell xenografts dosedon day 0 with: temozolomide 100 mg/kg orally daily for 5 days, FormulaIb at 10 mg/kg orally once weekly for 3 weeks, Formula Ib at 5 mg/kgorally weekly for 3 weeks, and the combinations of: Formula Ib at 10mg/kg orally once weekly for 3 weeks and temozolomide 100 mg/kg orallydaily for 5 days; and Formula Ib at 5 mg/kg orally once weekly for 3weeks and temozolomide 100 mg/kg orally daily for 5 days, along withmice receiving no drug (Vehicle group).

FIG. 45 shows the mean tumor volume change over time with Harlan femalenu/nu mice with SKOV3 human ovarian cancer tumor cell xenografts dosedon day 0 with: Formula Ia (GDC-0941) 73 mg/kg orally daily for 3 weeks,Formula Ia 36 mg/kg orally daily for 3 weeks, docetaxel 10 mg/kgintraperitoneal weekly for 3 weeks, and the combinations of Formula Ia73 mg/kg orally daily for 3 weeks and docetaxel 10 mg/kg intravenouslyweekly for 3 weeks; Formula Ia 36 mg/kg orally daily for 3 weeks anddocetaxel 10 mg/kg intravenously weekly for 3 weeks; and Formula Ia 73mg/kg orally weekly for 3 weeks and docetaxel 10 mg/kg intravenouslyweekly for 3 weeks, along with mice receiving no drug (Vehicle group).

FIG. 46 shows the mean tumor volume change over time with Harlan femalenu/nu mice with SKOV3 human ovarian cancer tumor cell xenografts dosedon day 0 with: Formula Ib 5 mg/kg orally daily for 3 weeks, Formula Ib 1mg/kg orally daily for 3 weeks, docetaxel 10 mg/kg intravenously weeklyfor 3 weeks, and the combinations of: Formula Ib 5 mg/kg orally dailyfor 3 weeks and docetaxel 10 mg/kg intravenously weekly for 3 weeks; andFormula Ib 1 mg/kg orally daily for 3 weeks and docetaxel 10 mg/kgintravenously weekly for 3 weeks; along with mice receiving no drug(Vehicle group).

FIG. 47 shows the mean tumor volume change over time with Harlan femalenu/nu mice with SKOV3 human ovarian cancer tumor cell xenografts dosedon day 0 with: Formula Ib 5 mg/kg orally weekly for 3 weeks, Formula Ib10 mg/kg orally weekly for 3 weeks, docetaxel 10 mg/kg intravenouslyweekly for 3 weeks, and the combinations of: Formula Ib 5 mg/kg orallyweekly for 3 weeks and docetaxel 10 mg/kg intravenously weekly for 3weeks; and Formula Ib 10 mg/kg orally weekly for 3 weeks and docetaxel10 mg/kg intravenously weekly for 3 weeks, along with mice receiving nodrug (Vehicle group).

FIG. 48 shows the mean tumor volume change over time with female SCIDBeige nude mice with LuCap 35V human primary prostate cancer tumor cellxenografts dosed on day 0 with: docetaxel 5 mg/kg intravenously on days1, 5 and 9 (q4d×3), Formula Ia (GDC-0941) 50 mg/kg orally daily for 18days, Formula Ia 100 mg/kg orally daily for 18 days, and thecombinations of: docetaxel 5 mg/kg intravenously on days 1, 5 and 9(q4d×3) and Formula Ia 50 mg/kg orally daily for 18 days and docetaxel 5mg/kg intravenously on days 1, 5 and 9 (q4d×3) and Formula Ia 100 mg/kgorally daily for 18 days, along with mice receiving no drug (Vehiclegroup).

FIG. 49 shows the mean tumor volume change over time with female SCIDBeige nude mice with LuCap 35V human primary prostate cancer tumor cellxenografts dosed on day 0 with: docetaxel 5 mg/kg intravenously on days1, 5 and 9 (q4d×3), Formula Ib 2.5 mg/kg orally daily for 15 days,Formula Ib 5 mg/kg orally daily for 15 days, and the combinations of:docetaxel 5 mg/kg intravenously on days 1, 5 and 9 (q4d×3) and FormulaIb 2.5 mg/kg orally daily for 15 days and docetaxel 5 mg/kgintravenously on days 1, 5 and 9 (q4d×3) and Formula Ib 5 mg/kg orallydaily for 15 days, along with mice receiving no drug (Vehicle group).

FIG. 50 shows the mean tumor volume change over time with CRL femalenu/nu mice with PC3-NCI human primary prostate cancer tumor cellxenografts dosed on days 1, 5, 9, and 13 (q4d×4) with: docetaxel 2.5mg/kg intravenously, Formula Ib 10 mg/kg orally on days 1, 5, 9 and 13(q4d×4), and the combination of: docetaxel 2.5 mg/kg intravenously andFormula Ib 10 mg/kg orally on days 1, 5, 9 and 13 (q4d×4), along withmice receiving no drug (Vehicle group).

FIG. 51 shows the mean tumor volume change over time with CRL femalenu/nu mice with PC3-NCI human primary prostate cancer tumor cellxenografts dosed on day 0 with: gemcitabine 100 mg/kg intraperitonealevery 3 days for 4 times, Formula Ia (GDC-0941) 150 mg/kg orally every 3days (q3d) for 4 times, Formula Ib 2.5 mg/kg orally every 3 days (q3d)for 4 times, Formula Ib 5 mg/kg orally every 3 days for 4 times, and thecombinations of: gemcitabine 100 mg/kg intraperitoneal every 3 days for4 times and Formula Ia 150 mg/kg orally every 3 days for 4 times;gemcitabine 100 mg/kg intraperitoneal every 3 days for 4 times andFormula Ib 2.5 mg/kg orally every 3 days for 4 times, gemcitabine 100mg/kg intraperitoneal every 3 days for 4 times and Formula Ib 5 mg/kgorally every 3 days for 4 times and Formula Ib 10 mg/kg orally every 3days for 4 times, along with mice receiving no drug (Vehicle group).

FIG. 52 shows the mean tumor volume change over time with Harlan femalenude mice with NCI-H2122 (K-ras) NSCLC tumor cell xenografts dosed onday 0 with: PD-0325901 6.3 mg/kg orally daily for 21 days, Formula Ia(GDC-0941) at 100 mg/kg orally daily for 21 days, and the combinationof: PD-0325901 6.3 mg/kg orally daily for 21 days and Formula Ia at 100mg/kg orally daily for 21 days, along with mice receiving no drug(Vehicle group).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.

Definitions

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and claims are intended tospecify the presence of stated features, integers, components, or steps,but they do not preclude the presence or addition of one or more otherfeatures, integers, components, steps, or groups thereof.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms, wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. Examples ofalkyl groups include, but are not limited to, methyl (Me, —CH₃), ethyl(Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimemthyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to twelve carbon atoms with at least one siteof unsaturation, i.e., a carbon-carbon, sp² double bond, wherein thealkenyl radical may be optionally substituted independently with one ormore substituents described herein, and includes radicals having “cis”and “trans” orientations, or alternatively, “E” and “Z” orientations.Examples include, but are not limited to, ethylenyl or vinyl (—CH═CH₂),allyl (—CH₂CH═CH₂), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to twelve carbon atoms with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms as a monocyclic ring or 7to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to12 atoms can be arranged, for example, as a bicyclo [4,5], [5,5], [5,6]or [6,6] system, and bicyclic carbocycles having 9 or 10 ring atoms canbe arranged as a bicyclo [5,6] or [6,6] system, or as bridged systemssuch as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Some aryl groups are representedin the exemplary structures as “Ar”. Aryl includes bicyclic radicalscomprising an aromatic ring fused to a saturated, partially unsaturatedring, or aromatic carbocyclic or heterocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.Aryl groups are optionally substituted independently with one or moresubstituents described herein.

The terms “heterocycle,” “hetercyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 20 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen and sulfur,the remaining ring atoms being C, where one or more ring atoms isoptionally substituted independently with one or more substituentsdescribed below. A heterocycle may be a monocycle having 3 to 7 ringmembers (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O,P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atomsand 1 to 6 heteroatoms selected from N, O, P, and S), for example: abicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocycles are describedin Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W.A.Benjamin, N.Y., 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “TheChemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley& Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. The term “heterocycle”includes heterocycloalkoxy. “Heterocyclyl” also includes radicals whereheterocycle radicals are fused with a saturated, partially unsaturatedring, or aromatic carbocyclic or heterocyclic ring. Examples ofheterocyclic rings include, but are not limited to, pyrrolidinyl,tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl,dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino,thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl,oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl,diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl,2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl,dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring carbon atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl,benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.Heteroaryl groups are optionally substituted independently with one ormore substituents described herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked),nitrogen (nitrogen-linked) or oxygen (oxygen-linked) attached where suchis possible. By way of example and not limitation, carbon bondedheterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6 of apyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4,or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole ortetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole orthiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole,position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine,position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5,6, 7, or 8 of an isoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

“Carbon linked monocyclic heteroaryl” refers to a five- or six-membered,unsubstituted or substituted, monocyclic heteroaryl radical whichcontains 1, 2, 3 or 4 ring heteroatoms independently selected from N, Oand S. The carbon linked monocyclic heteroaryl is attached to the C-2position of the pyrimidine ring according to Formulas I and II at anycarbon atom of the monocyclic heteroaryl R³ group. Carbon linkedmonocyclic heteroaryl radicals include, but are not limited to:2-pyridyl, 3-pyridyl, 4-pyridyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-imidazolyl, 4-imidazolyl, 3-pyrazolyl, 4-pyrazolyl,2-pyrrolyl, 3-pyrrolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 2-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 2-oxazolyl, 4-oxazolyl,5-oxazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 3-triazolyl,1-triazolyl, 5-tetrazolyl, 1-tetrazolyl, and 2-tetrazolyl. Carbon linkedmonocyclic heteroaryls are optionally substituted independently with oneor more substituents described herein.

“Carbon linked fused bicyclic C₃-C₂₀ heterocyclyl” and “carbon linkedfused bicyclic C₁-C₂₀ heteroaryl” containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, differ only bytheir aromatic character, and have two rings fused together, i.e. sharea common bond. Carbon linked fused bicyclic heterocyclyl and heteroarylradicals are attached to the C-2 position of the pyrimidine ringaccording to Formulas I and II at any carbon atom of the fused bicyclicC₃-C₂₀ heterocyclyl or fused bicyclic C₁-C₂₀ heteroaryl group R³ group.Carbon linked fused bicyclic heterocyclyl and heteroaryl radicalsinclude, but are not limited to: 1H-indazole, 1H-indole, indolin-2-one,1-(indolin-1-yl)ethanone, 1H-benzo[d][1,2,3]triazole,1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[3,4-d]pyrimidine,1H-benzo[d]imidazole, 1H-benzo[d]imidazol-2(3H)-one,1H-pyrazolo[3,4-c]pyridine, 1H-pyrrolo[2,3-c]pyridine,3H-imidazo[4,5-c]pyridine, 7H-pyrrolo[2,3-d]pyrimidine, 7H-purine,1H-pyrazolo[4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine,2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline,isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one,3,4-dihydroquinolin-2(1H)-one, quinazolin-2(1H)-one,quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine, andpyrido[3,2-b]pyrazine. Fused bicyclic heterocycles and fused bicyclicheteroaryls are optionally substituted independently with one or moresubstituents described herein.

The substituent groups that alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, fused bicyclic C₄-C₂₀ heterocyclyl, andfused bicyclic C₁-C₂₀ heteroaryl are optionally substituted with includeF, Cl, Br, I, CN, CF₃, —NO₂, oxo, R¹⁰, —C(═Y)R¹⁰, —C(═Y)OR¹⁰,—C(═Y)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(n)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(n)OR¹⁰, —NR¹⁰R¹¹,—NR¹²C(═Y)R¹⁰, —NR¹², C(═Y)OR¹¹, —NR¹²C(═Y)NR¹⁰R¹¹, —NR¹²SO₂R¹⁰, ═NR¹²,OR¹⁰, —OC(═Y)R¹⁰, —OC(═Y)OR¹⁰, —OC(═Y)NR¹⁰R¹¹, —OS(O)₂(OR¹⁰),—OP(═Y)(OR¹⁰)(OR¹¹), —OP(OR¹⁰(OR¹¹), SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰,—S(O)₂NR¹⁰R¹¹, —S(O)(OR¹⁰), —S(O)₂(OR¹⁰), —SC(═Y)R¹⁰, —SC(═Y)OR¹⁰,—SC(═Y)NR¹⁰R¹¹, C₁-C₁₂ optionally substituted alkyl, C₂-C₈ optionallysubstituted alkenyl, C₂-C₈ optionally substituted alkynyl, C₃-C₁₂optionally substituted carbocyclyl, C₂-C₂₀ optionally substitutedheterocyclyl, C₆-C₂₀ optionally substituted aryl, C₁-C₂₀ optionallysubstituted heteroaryl, —(CR¹⁴R¹⁵)_(t)—NR¹²C(═O)(CR¹⁴R¹⁵)NR¹⁰R¹¹, and(CR⁴R⁵)_(t)—NR¹⁰R¹¹

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the growth, development or spread of cancer. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) prevents or delays the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small- cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

A “chemotherapeutic agent” is a biological (large molecule) or chemical(small molecule) compound useful in the treatment of cancer, regardlessof mechanism of action. Classes of chemotherapeutic agents include, butare not limited to: alkylating agents, antimetabolites, spindle poisonplant alkaloids, cytotoxic/antitumor antibiotics, topoisomeraseinhibitors, proteins, antibodies, photosensitizers, and kinaseinhibitors. Chemotherapeutic agents include compounds used in “targetedtherapy” and non-targeted conventional chemotherapy.

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), bevacizumab (AVASTIN®, Genentech), trastuzumab(HERCEPTIN®, Genentech), pertuzumab (OMNITARG®, rhuMab 2C4, Genentech),temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, rapamycin, and lapatinib (TYKERB®, Glaxo SmithKline).

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (MEK inhibitor, Exelixis, WO2007/044515), ARRY-886 (MEK inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), ABT-869(multi-targeted inhibitor of VEGF and PDGF family receptor tyrosinekinases, Abbott Laboratories and Genentech), ABT-263 (Bcl-2/Bcl-xLinhibitor, Abbott Laboratories and Genentech), PTK787/ZK 222584(Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinicacid), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib(NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca),irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson& Johnson), capecitabine (XELODA®, Roche), ABRAXANE™ (Cremophor-free),albumin-engineered nanoparticle formulations of paclitaxel (AmericanPharmaceutical Partners, Schaumberg, I1), vandetanib (rINN, ZD6474,ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen),temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline),canfosfamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide(CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gammalI, calicheamicin omegaI1, dynemicin,dynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids andderivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the PI3K inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

The term “mammal” includes, but is not limited to, humans, mice, rats,guinea pigs, monkeys, dogs, cats, horses, cows, pigs, and sheep, andpoultry.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate))salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.Acids which are generally considered suitable for the formation ofpharmaceutically useful or acceptable salts from basic pharmaceuticalcompounds are discussed, for example, by P. Stahl et al, Camille G.(eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use.(2002) Zurich: Wiley-VCH; S. Berge et al, Journal of PharmaceuticalSciences (1977) 66(1) 1 19; P. Gould, International J. of Pharmaceutics(1986) 33 201 217; Anderson et al, The Practice of Medicinal Chemistry(1996), Academic Press, New York; Remington's Pharmaceutical Sciences,18^(th) ed., (1995) Mack Publishing Co., Easton Pa.; and in The OrangeBook (Food & Drug Administration, Washington, D.C. on their website).These disclosures are incorporated herein by reference thereto.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

A “solvate” refers to a physical association or complex of one or moresolvent molecules and a compound of the invention. The compounds of theinvention may exist in unsolvated as well as solvated forms. Examples ofsolvents that form solvates include, but are not limited to, water,isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, andethanolamine. The term “hydrate” refers to the complex where the solventmolecule is water. This physical association involves varying degrees ofionic and covalent bonding, including hydrogen bonding. In certaininstances the solvate will be capable of isolation, for example when oneor more solvent molecules are incorporated in the crystal lattice of thecrystalline solid. Preparation of solvates is generally known, forexample, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601 611 (2004).Similar preparations of solvates, hemisolvate, hydrates and the like aredescribed by E. C. van Tonder et al, AAPS PharmSciTech., 5(1), article12 (2004); and A. L. Bingham et al, Chem. Commun., 603 604 (2001). Atypical, non-limiting, process involves dissolving the inventivecompound in desired amounts of the desired solvent (organic or water ormixtures thereof) at a higher than ambient temperature, and cooling thesolution at a rate sufficient to form crystals which are then isolatedby standard methods. Analytical techniques such as, for example I.R.spectroscopy, show the presence of the solvent (or water) in thecrystals as a solvate (or hydrate).

The term “synergistic” as used herein refers to a therapeuticcombination which is more effective than the additive effects of the twoor more single agents. A determination of a synergistic interactionbetween a Formula I or II compound, and one or more chemotherapeuticagent may be based on the results obtained from the assays describedherein. The results of these assays are analyzed using the Chou andTalalay combination method and Dose-Effect Analysis with CalcuSynsoftware in order to obtain a Combination Index (Chou and Talalay, 1984,Adv. Enzyme Regul. 22:27-55). The combinations provided by thisinvention have been evaluated in several assay systems, and the data canbe analyzed utilizing a standard program for quantifying synergism,additivism, and antagonism among anticancer agents. The programpreferably utilized is that described by Chou and Talalay, in “NewAvenues in Developmental Cancer Chemotherapy,” Academic Press, 1987,Chapter 2. Combination Index values less than 0.8 indicates synergy,values greater than 1.2 indicate antagonism and values between 0.8 to1.2 indicate additive effects. The combination therapy may provide“synergy” and prove “synergistic”, i.e., the effect achieved when theactive ingredients used together is greater than the sum of the effectsthat results from using the compounds separately. A synergistic effectmay be attained when the active ingredients are: (1) co-formulated andadministered or delivered simultaneously in a combined, unit dosageformulation; (2) delivered by alternation or in parallel as separateformulations; or (3) by some other regimen. When delivered inalternation therapy, a synergistic effect may be attained when thecompounds are administered or delivered sequentially, e.g., by differentinjections in separate syringes. In general, during alternation therapy,an effective dosage of each active ingredient is administeredsequentially, i.e., serially, whereas in combination therapy, effectivedosages of two or more active ingredients are administered together.

Formula I and II Compounds

The present invention includes therapeutic combinations includingFormula I and II compounds which have the structures:

or stereoisomers, geometric isomers, tautomers, or pharmaceuticallyacceptable salts thereof, where:

R¹ is selected from H, F, Cl, Br, I, CN, —(CR¹⁴R¹⁵)_(m)NR¹⁰R¹¹,C(R¹⁴R¹⁵)_(n)NR¹²C(═Y)R¹⁰, —(CR¹⁴R¹⁵)_(n)NR¹²S(O)₂R¹⁰,—(CR¹⁴R¹⁵)_(m)OR¹⁰, —(CR¹⁴R¹⁵)_(n)S(O)₂R¹⁰, —(CR¹⁴R¹⁵)_(n)S(O)₂NR¹⁰R¹¹,—C(OR¹⁰)R¹¹R¹⁴, —C(═Y)R¹⁰, —C(═Y)OR¹⁰, —C(═Y)NR¹⁰R¹¹, —C(═Y)NR¹²OR¹⁰,—C(═O)NR¹²S(O)₂R¹⁰, —C(═O)NR¹²(CR¹⁴R¹⁵)_(m)NR¹⁰R¹¹, —NO₂, —NR¹²C(═Y)R¹¹,—NR¹²C(═Y)OR¹¹, —NR¹²C(═Y)NR¹⁰R¹¹, —NR¹²S(O)₂R¹⁰, —NR¹²SO₂NR¹⁰R¹¹,—SR¹⁰, —S(O)₂R¹⁰, —S(O)₂NR¹⁰R¹¹, —SC(═Y)R¹⁰, —SC(═Y)OR¹⁰, C₁-C₁₂ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl,C₆-C₂₀ aryl, and C₁-C₂₀ heteroaryl;

R² is selected from H, F, Cl, Br, I, CN, CF₃, —NO₂, —C(═Y)R¹⁰,—C(═Y)OR¹⁰, —C(═Y)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(m)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(n)OR¹⁰,—(CR¹⁴R¹⁵)_(t)—NR¹²C(═O)(CR¹⁴R¹⁵)NR¹⁰R¹¹, —NR¹²C(═Y)R¹⁰, —NR¹²C(═Y)OR¹⁰,—NR¹²C(═Y)NR¹⁰R¹¹, —NR¹²SO₂R¹⁰, OR¹⁰, —OC(═Y)R¹⁰, —OC(═Y)OR¹⁰,—OC(═Y)NR¹⁰R¹¹, —OS(O)₂(OR¹⁰), —OP(═Y)(OR¹⁰)(OR¹¹), —OP(OR10(OR¹¹),SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —S(O)₂NR¹⁰R¹¹, —S(O)(OR¹⁰), —S(O)₂(OR¹⁰),—SC(═Y)R¹⁰, —SC(═Y)OR¹⁰, —SC(═Y)NR¹⁰R¹¹, C₁-C₁₂ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl;

R³ is a carbon linked monocyclic heteroaryl, a carbon linked fusedbicyclic C₃-C₂₀ heterocyclyl, or a carbon linked fused bicyclic C₁-C₂₀heteroaryl, where the monocyclic heteroaryl, fused bicyclic C₃-C₂₀heterocyclyl, and fused bicyclic C₁-C₂₀ heteroaryl are optionallysubstituted with one or more groups selected from F, Cl, Br, I, —CN,—NR¹⁰R¹¹, —OR¹⁰, —C(O)R¹⁰, —NR¹⁰C(O)R¹¹, —N(C(O)R¹¹)₂, —NR¹⁰C(O)NR¹⁰R¹¹,—NR¹²S(O)₂R¹⁰, —C(═O)OR¹⁰, —C(═O)NR¹⁰R¹¹, C₁-C₁₂ alkyl and (C₁-C₁₂alkyl)-OR¹⁰;

R¹⁰, R¹¹ and R¹² are independently H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, or C₁-C₂₀heteroaryl,

or R¹⁰ and R¹¹ together with the nitrogen to which they are attachedform a C₂-C₂₀ heterocyclic ring optionally substituted with one or moregroups independently selected from oxo, (CH₂)_(m)OR¹², NR¹²R¹², CF₃, F,Cl, Br, I, SO₂R¹², C(═O)R¹², NR¹²C(═Y)R¹², NR¹²S(O)₂R¹², C(═Y)NR¹²R¹²,C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀heterocyclyl, C₆-C₂₀ aryl and C₁-C₂₀ heteroaryl;

R¹⁴ and R¹⁵ are independently selected from H, C₁-C₁₂ alkyl, or—(CH₂)_(n)-aryl,

or R¹⁴ and R¹⁵ together with the atoms to which they are attached form asaturated or partially unsaturated C₃-C₁₂ carbocyclic ring;

where said alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl, are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, CN, CF₃, —NO₂, oxo, R¹⁰,—C(═Y)R¹⁰, —C(═Y)OR¹⁰, —C(═Y)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(n)NR¹⁰R¹¹,—(CR¹⁴R¹⁵)_(n)OR¹⁰, —NR¹⁰R¹¹, —NR¹²C(═Y)OR¹¹, —NR¹²C(═Y)NR¹⁰R¹¹,—(CR¹⁴R¹⁵)_(m)NR¹²SO₂R¹⁰, ═NR¹², OR¹⁰, —OC(═Y)R¹⁰, —OC(═Y)OR¹⁰,—OC(═Y)NR¹⁰R¹¹, —OS(O)₂(OR¹⁰), —OP(═Y)(OR¹⁰(OR¹¹), —OP(OR¹⁰)(OR¹¹),—SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —S(O)₂NR¹⁰R¹¹, —S(O)(OR¹⁰), —S(O)₂(OR¹⁰),—SC(═Y)R¹⁰, —SC(═Y)OR¹⁰, —SC(═Y)NR¹⁰R¹¹, C₁-C,₂ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl;

Y is O, S, or NR¹²;

m is 0, 1, 2, 3, 4, 5 or 6; and

n is 1, 2, 3, 4, 5 or 6.

Exemplary embodiments of Formula I and II compounds include wherein R¹is —(CR¹⁴R¹⁵)_(m)NR¹⁰R¹¹ where m is 1, and R¹⁰ and R¹¹ together with thenitrogen to which they are attached form an optionally substitutedC₃-C₂₀ heterocyclic ring. The C₃-C₂₀ heterocyclic ring may bepiperazinyl, optionally substituted with one or more groups selectedfrom NR¹⁰R¹¹, CF₃, F, Cl, Br, I, SO₂R¹⁰, C(═O)R¹⁰, NR¹²C(═Y)R¹¹,NR¹²S(O)₂R¹¹, C(═Y)NR¹⁰R¹¹, and C₁-C₁₂ alkyl.

Exemplary embodiments of Formula I and II compounds include wherein R¹is not H.

Exemplary embodiments of Formula I and II compounds include wherein R²is H, CH₃, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, orC₁-C₂₀ heteroaryl. The C₁-C₂₀ heteroaryl may be a monocyclic heteroarylgroup selected from 2-pyridyl, 3-pyridyl, 4-pyridyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-imidazolyl, 4-imidazolyl, 3-pyrazolyl,4-pyrazolyl, 2-pyrrolyl, 3-pyrrolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 2-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 2-oxazolyl, 4-oxazolyl,5-oxazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 3-triazolyl,1-triazolyl, 5-tetrazolyl, 1-tetrazolyl, and 2-tetrazolyl.

Exemplary embodiments of Formula I and II compounds include wherein R³is 2-aminopyrimidin-5-yl.

Exemplary embodiments of Formula I and II compounds include wherein R³is a bicyclic heteroaryl group selected from 1H-indazole, 1H-indole,indolin-2-one, 1-(indolin-1-yl)ethanone, 1H-benzo[d][1,2,3]triazole,1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[3,4-d]pyrimidine,1H-benzo[d]imidazole, 1H-benzo[d]imidazol-2(3H)-one,1H-pyrazolo[3,4-c]pyridine, 1H-pyrrolo[2,3-c]pyridine,3H-imidazo[4,5-c]pyridine, 7H-pyrrolo[2,3-d]pyrimidine, 7H-purine,1H-pyrazolo[4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine,2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline,isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one,3,4-dihydroquinolin-2(1H)-one, quinazolin-2(1H)-one,quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine, andpyrido[3,2-b]pyrazine.

Exemplary embodiments of Formula I and II compounds include wherein R³is 1H-indazol-4-yl.

An exemplary Formula I compound is4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholinehaving Formula Ia:

Another exemplary Formula I compound is(S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-onehaving Formula Ib:

An exemplary Formula II compound is4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholinehaving Formula IIa:

Preparation of Formula I and II Compounds

The Formula I and II compounds may be synthesized by synthetic routesthat include processes analogous to those well-known in the chemicalarts, and including WO 2006/046031. Starting materials are generallyavailable from commercial sources such as Aldrich Chemicals (Milwaukee,Wis.) or are readily prepared using methods well known to those skilledin the art (e.g., prepared by methods generally described in Louis F.Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley,N.Y. (1967-1999 ed.), or Beilsteins Handbuch der organischen Chemie, 4,Aufl. ed. Springer-Verlag, Berlin, including supplements (also availablevia the Beilstein online database).

Formula I and II compound may be prepared using procedures to prepareother thiophenes and pyrimidines (U.S. Pat. Nos. 6,608,053; 6,492,383;6,232,320; 6,187,777; 3,763,156; 3,661,908; 3,475,429; 5,075,305; US2003/220365; GB 1393161; WO 93/13664;); and other heterocycles, whichare described in: Comprehensive Heterocyclic Chemistry, EditorsKatritzky and Rees, Pergamon Press, 1984.

Formula I and II compounds may be converted into a pharmaceuticallyacceptable salt, and a salt may be converted into the free basecompound, by conventional methods. Formula I and II compounds may betherapeutically effective as a free base or as a pharmaceuticallyacceptable salt, depending on the desired properties such as solubility,dissolution, hygroscopic nature, and pharmacokinetics. Examples ofpharmaceutically acceptable salts include salts with inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuricacid, nitric acid and phosphoric acid; and organic acids such asmethanesulfonic acid, benzenesulphonic acid, formic acid, acetic acid,trifluoroacetic acid, propionic acid, oxalic acid, malonic acid,succinic acid, fumaric acid, maleic acid, lactic acid, malic acid,tartaric acid, citric acid, ethanesulfonic acid, aspartic acid andglutamic acid. The salt may be a mesylate, a hydrochloride, a phosphate,a benzenesulfonate or a sulfate. Salts may be mono-salts or bis-salts.For example, the mesylate salt may be the mono-mesylate or thebis-mesylate.

Formula I and II compounds and salts may also exist as hydrates orsolvates.

Protection of functional groups (e.g., primary or secondary amine) ofintermediates may be necessary in preparing Formula I and II compounds.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, 1991.

For illustrative purposes, Schemes 1-7 show general methods forpreparing the compounds of the present invention as well as keyintermediates. For a more detailed description of the individualreaction steps, see the Examples section below. Those skilled in the artwill appreciate that other synthetic routes may be used to synthesizethe inventive compounds. Although specific starting materials andreagents are depicted in the Schemes and discussed below, other startingmaterials and reagents can be easily substituted to provide a variety ofderivatives and/or reaction conditions. In addition, many of thecompounds prepared by the methods described below can be furthermodified in light of this disclosure using conventional chemistry wellknown to those skilled in the art.

Scheme 1 shows a general method for preparation of the thienopyrimidineintermediates 55 and 56 from 2-carboxyester, 3-amino thiophene, and2-amino, 3-carboxy ester thiophene reagents, respectively 51 and 52,wherein Hal is Cl, Br, or I; and R¹, R², and R¹⁰ are as defined forFormula I and II compounds, or precursors or prodrugs thereto.

Scheme 2 shows a general method for selectively displacing a 4-halidefrom bis-halo thienopyrimidine intermediates 57 and 58 with morpholineunder basic conditions in an organic solvent to prepare 2-halo,4-morpholino thienopyrimidine compounds 59 and 60 respectively, whereinHal is Cl, Br, or I; and R¹ and R² are as defined for Formula I and IIcompounds, or precursors or prodrugs thereto.

Scheme 3 shows a general method for derivatizing the 6-position of2-halo, 4-morpholino, 6-hydrogen thienopyrimidine compounds 61 and 62where R¹ is H. Treating 61 or 62 with a lithiating reagent to remove the6 position proton, followed by adding an acylating reagent R¹⁰C(O)Zwhere Z is a leaving group, such as halide, NHS ester, carboxylate, ordialkylamino, gives 2-halo, 4-morpholino, 6-acyl thienopyrimidinecompounds 63 and 64, wherein Hal is Cl, Br, or I; and R² and R¹⁰ are asdefined for Formula I and II compounds, or precursors or prodrugsthereto. An example of R¹⁰C(O)Z to prepare 6-formyl compounds (R¹⁰′H) isN,N′-dimethylformamide (DMF).

Scheme 4 shows a general method for Suzuki-type coupling of a 2-halopyrimidine intermediate (65 and 66) with a monocyclic heteroaryl, fusedbicyclic heterocyclyl or fused bicyclic heteroaryl boronate acid (R¹⁵═H)or ester (R¹⁵═alkyl) reagent 67 to prepare the 2-substituted (Hy),4-morpholino thienopyrimidine compounds (68 and 69) of Formulas I and IIwherein Hal is Cl, Br, or I; and R¹ and R² are as defined for Formula Iand II compounds, or precursors or prodrugs thereto. For reviews of theSuzuki reaction, see: Miyaura et al. (1995) Chem. Rev. 95:2457-2483;Suzuki, A. (1999) J. Organomet. Chem. 576:147-168; Suzuki, A. inMetal-Catalyzed Cross-Coupling Reactions, Diederich, F., Stang, P. J.,Eds., VCH, Weinheim, Del. (1998), pp 49-97. The palladium catalyst maybe any that is typically used for Suzuki-type cross-couplings, such asPdCl₂(PPh₃)₂, Pd(PPh₃)₄, Pd(OAc)₂, PdCl₂(dppf)-DCM, Pd₂(dba)₃/Pt-Bu)₃(Owens et al (2003) Bioorganic & Med. Chem. Letters 13:4143-4145;Molander et al (2002) Organic Letters 4(11):1867-1870; U.S. Pat. No.6,448,433).

Scheme 5 shows a general method for the synthesis of alkynes 71, whichcan be used to prepare alkynylated derivatives of compounds 72 and 73.Propargylic amines 71 may be prepared by reaction of propargyl bromide70 with an amine of the formula R¹⁰R¹¹NH (wherein R¹⁰ and R¹¹ areindependently selected from H, alkyl, aryl and heteroaryl, or R¹⁰ andR¹¹ together with the nitrogen to which they are attached form aheterocyclic ring) in the presence of an appropriate base (Cs₂CO₃ or thelike). For reviews of alkynyl amines and related syntheses seeBooker-Milburn, K. I., Comprehensive Organic Functional GroupTransformations (1995), 2:1039-1074; and Viehe, H. G., (1967) Angew.Chem., Int. Ed. Eng., 6(9):767-778. Alkynes 71 may subsequently bereacted with intermediates 72 (X²=bromo or iodo) or 73 (via Sonogashiracoupling), to provide compounds 74 and 75, respectively, wherein R² andR³ are as defined for Formula I and II compounds, or precursors orprodrugs thereto.

Scheme 6 shows a general method for the synthesis of alkynes 77, whichcan be used to prepare alkynylated derivatives of compounds 72 and 73.Gem-dialkyl propargylic amines 77 may be prepared using methodsdescribed by Zaragoza et al (2004) J. Med. Chem., 47:2833. According toScheme 6, gem-dialkyl chloride 76 (R¹⁴ and R¹⁵ are independently methyl,ethyl or other alkyl group) can be reacted with an amine of the formulaR¹⁰R¹¹NH (wherein R¹⁰ and R¹¹ are independently selected from H, alkyl,aryl and heteroaryl, or R¹⁰ and R¹¹ together with the nitrogen to whichthey are attached form a heterocyclic ring) in the presence of CuCl andan appropriate base (e.g. TEA or the like) to provide the alkyne 77.Alkyne 77 can be reacted with intermediates 72 or 73 (via Sonogashiracoupling) to provide compounds 78 and 79, respectively, wherein R² andR³ are as defined for Formula I and II compounds, or precursors orprodrugs thereto.

Scheme 7 shows a general scheme for the synthesis of alkynes 81, whichcan be used to prepare alkynylated derivatives of compounds 72 and 73.But-3-yn-1-amines 81 (wherein R¹⁴ and R¹⁵ are independently H, alkyl,aryl, heteroaryl, or R¹⁴ and R¹⁵ together with the carbon atom to whichthey are attached form a carbocyclic or heterocyclic ring) can beprepared from reaction of alkynes 80 (LG=tosylate or other leavinggroup) with an amine of the formula R¹⁰R¹¹NH (wherein R¹⁰ and R¹¹ areindependently selected from H, alkyl, aryl and heteroaryl, or R¹⁰ andR¹¹ together with the nitrogen to which they are attached form aheterocyclic ring) using the protocol described by Olomucki M. et al(1960) Ann. Chim. 5:845. Alkynes 81 can subsequently be reacted withintermediates 72 or 73 (via Sonogashira coupling), according to thedescriptions provided for Schemes 5 and 6 to provide compounds 82 and83, respectively, wherein R² and R³ are as defined for Formula I and IIcompounds, or precursors or prodrugs thereto.

A pharmaceutically acceptable salt of a thienopyrimidine compound ofFormula I or II may be prepared using conventional techniques. Typicallythe process comprises treating the thienopyrimidine of Formula I asdefined above with a suitable acid in a suitable solvent.

In the process of the invention as defined above, both the aminationstep and the Pd-mediated cross-coupling step take place underconventional conditions. The palladium catalyst may be any that istypically used for Suzuki-type cross-couplings, such as PdCl₂(PPh₃)₂.The reducing agent is typically a borohydride, such as NaBH(OAc)₃, NaBH₄or NaCNBH₄.

Methods of Separation

In the methods of preparing the compounds of this invention, it may beadvantageous to separate reaction products from one another and/or fromstarting materials. The desired products of each step or series of stepsis separated and/or purified (hereinafter separated) to the desireddegree of homogeneity by the techniques common in the art. Typicallysuch separations involve multiphase extraction, crystallization from asolvent or solvent mixture, distillation, sublimation, orchromatography. Chromatography can involve any number of methodsincluding, for example: reverse-phase and normal phase; size exclusion;ion exchange; high, medium and low pressure liquid chromatographymethods and apparatus; small scale analytical; simulated moving bed(SMB) and preparative thin or thick layer chromatography, as well astechniques of small scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved. For example, boiling point and molecular weightin distillation and sublimation, presence or absence of polar functionalgroups in chromatography, stability of materials in acidic and basicmedia in multiphase extraction, and the like. One skilled in the artwill apply techniques most likely to achieve the desired separation.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisqmer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine(amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.,(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

Chemotherapeutic Agents

Certain chemotherapeutic agents have demonstrated surprising andunexpected properties in combination with Formula I or II compounds ininhibiting cellular proliferation in vitro and in vivo. Suchchemotherapeutic agents include: erlotinib, docetaxel, 5-FU,gemcitabine, PD-0325901, cisplatin, carboplatin, paclitaxel,bevacizumab, trastuzumab, pertuzumab, temozolomide, tamoxifen,doxorubicin, Akti-1/2, HPPD, and rapamycin.

Erlotinib (TARCEVA®, OSI-774, Genentech) is used to treat non-small celllung cancer (NSCLC), lung cancer, pancreatic cancer and several othertypes of cancer by specifically targeting the epidermal growth factorreceptor (EGFR) tyrosine kinase (U.S. Pat. No. 5,747,498; U.S. Pat. No.6,900,221; Moyer et al (1997) Cancer Res. 57:4838; Pollack et al (1999)J. Pharmcol. Exp. Ther. 291:739; Perez-Soler et al (2004) J. Clin.Oncol. 22:3238; Kim et al (2002) Curr. Opin. Invest. Drugs 3:1385-1395;Blackhall et al (2005) Expert Opin. Pharmacother. 6:995-1002). Erlotinibis named asN-(3-ethynylphenyl)-6,7-bis(methoxymethoxy)quinazolin-4-amine (CAS Reg.No. 183321-74-6) and has the structure:

Docetaxel (TAXOTERE®, Sanofi-Aventis) is used to treat breast, ovarian,and NSCLC cancers (U.S. Pat. No. 4,814,470; U.S. Pat. No. 5,438,072;U.S. Pat. No. 5,698,582; U.S. Pat. No. 5,714,512; U.S. Pat. No.5,750,561; Mangatal et al (1989) Tetrahedron 45:4177; Ringel et al(1991) J. Natl. Cancer Inst. 83:288; Bissery et al (1991) Cancer Res.51:4845; Herbst et al (2003) Cancer Treat. Rev. 29:407-415; Davies et al(2003) Expert. Opin. Pharmacother. 4:553-565). Docetaxel is named as(2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with5, 20-epoxy-1,2,4,7,10,13-hexahydroxytax-11-en-9-one 4-acetate2-benzoate, trihydrate (U.S. Pat. No. 4,814,470; EP 253738; CAS Reg. No.114977-28-5) and has the structure:

5-FU (fluorouracil, 5-fluorouracil, CAS Reg. No. 51-21-8) is athymidylate synthase inhibitor and has been used for decades in thetreatment of cancer, including colorectal and pancreatic cancer (U.S.Pat. No. 2,802,005; U.S. Pat. No. 2,885,396; Duschinsky et al (1957) J.Am. chem. Soc. 79:4559; Hansen, R. M. (1991) Cancer Invest. 9:637-642).5-FU is named as 5-fluoro-1H-pyrimidine-2,4-dione, and has thestructure:

Gemcitabine (GEMZAR®, Lilly, CAS Reg. No. 95058-81-4) is a nucleosideanalog which blocks DNA replication, is used to treat various carcinomasincluding pancreatic, breast, NSCLC, and lymphomas (U.S. Pat. No.4,808,614; U.S. Pat. No. 5,464,826; Hertel et al (1988) J. Org. Chem.53:2406; Hertel et al (1990) Cancer Res. 50:4417; Lund et al (1993)Cancer Treat. Rev. 19:45-55). Gemcitabine is named as4-amino-1-[3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-pyrimidin-2-one,and has the structure:

PD-0325901 (CAS RN 391210-10-9, Pfizer) is a second-generation, non-ATPcompetitive, allosteric MEK inhibitor for the potential oral tablettreatment of cancer (U.S. Pat. Nos. 6,960,614; 6,972,298; US2004/147478; US 2005/085550). Phase II clinical trials have beenconducted for the potential treatment of breast tumors, colon tumors,and melanoma. PD-0325901 is named as(R)—N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide,and has the structure:

Cisplatin (cis-diamine, dichloroplatinum(II), CAS Reg. No. 15663-27-1),is a platinum-based chemotherapy drug used to treat various types ofcancers, including sarcomas, some carcinomas (e.g. small cell lungcancer and ovarian cancer), lymphomas and germ cell tumors (Ochs et al(1978) Cancer Treat. Rep. 62:239; Pinedo et al (1978) Eur. J. Cancer14:1149; “Cisplatin, Current Status and New Developments”, A. W.Prestayko et al., Eds., Academic Press, New York, 1980). Cisplatin(CDDP) was the first member of its class, which now also includescarboplatin and oxaliplatin.

Carboplatin (CAS Reg. No. 41575-94-4) is a chemotherapeutic drug usedagainst ovarian carcinoma, lung, head and neck cancers (U.S. Pat. No.4,140,707; Calvert et al (1982) Cancer Chemother. Pharmacol. 9:140;Harland et al (1984) Cancer Res. 44:1693). Carboplatin is named asazanide; cyclobutane-1,1-dicarboxylic acid; platinum, and has thestructure:

Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton N.J., CASReg. No. 33069-62-4) is isolated the compound from the bark of thePacific yew tree, Taxus brevifolia, and used to treat lung, ovarian,breast cancer, and advanced forms of Kaposi's sarcoma (Wani et al (1971)J. Am. Chem. Soc. 93:2325; Mekhail et al (2002) Expert. Opin.Pharmacother. 3:755-766). Paclitaxel is named asβ-(benzoylamino)-α-hydroxy-,6,12b-bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca(3,4)benz(1,2-b)oxet-9-ylester,(2aR-(2a-α,4-β,4a-β,6-β,9-α(α-R*,β-s*),11-α,12-α,12a-α,2b-α))-benzenepropanoicacid, and has the structure:

Bevacizumab (AVASTIN®, Genentech) is a recombinant humanized monoclonalantibody against VEGF, vascular endothelial growth factor (U.S. Pat. No.6,054,297; Presta et al (1997) Cancer Res. 57:4593-4599). It is used inthe treatment of cancer, where it inhibits tumor growth by blocking theformation of new blood vessels. Bevacizumab was the first clinicallyavailable angiogenesis inhibitor in the United States, approved by theFDA in 2004 for use in combination with standard chemotherapy in thetreatment of metastatic colon cancer and most forms of metastaticnon-small cell lung cancer. Several late-stage clinical studies areunderway to determine its safety and effectiveness for patients with:adjuvant/non-metastatic colon cancer, metastatic breast cancer,metastatic renal cell carcinoma, metastatic glioblastoma multiforme,metastatic ovarian cancer, metastatic hormone-refractory prostatecancer, and metastatic or unresectable locally advanced pancreaticcancer (Ferrara et al (2004) Nat. Rev. Drug Disc. 3:391-400).

Bevacizumab includes mutated human IgG1 framework regions andantigen-binding complementarity-determining regions from the murineanti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGFto its receptors. Bevacizumab has a molecular mass of about 149,000daltons and is glycosylated. Bevacizumab and other humanized anti-VEGFantibodies are further described in U.S. Pat. No. 6,884,879. Additionalanti-VEGF antibodies include the G6 or B20 series antibodies, e.g.,G6-31, B20-4.1, (WO 2005/012359; WO 2005/044853; U.S. Pat. Nos.7,060,269; 6,582,959; 6,703,020 ; 6,054,297; WO 98/45332; WO 96/30046;WO 94/10202; EP 0666868B1; US 2006/009360; US 2005/0186208; US2003/0206899; US 2003/0190317; US 2003/0203409; 20050112126; Popkov etal (2004) Journal of Immunological Methods 288:149-164. A “B20 seriesantibody” is an anti-VEGF antibody that is derived from a sequence ofthe B20 antibody or a B20-derived antibody according to any one of FIGS.27-29 of WO 2005/012359, the entire disclosure of which is expresslyincorporated herein by reference. In one embodiment, the B20 seriesantibody binds to a functional epitope on human VEGF comprising residuesF17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104. Other anti-VEGFantibodies include those that bind to a functional epitope on human VEGFcomprising residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, andC104 or, alternatively, comprising residues F17, Y21, Q22, Y25, D63, 183and Q89.

Trastuzumab (HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is arecombinant DNA-derived humanized, IgG1 kappa, monoclonal antibodyversion of the murine HER2 antibody which selectively binds with highaffinity in a cell-based assay (Kd=5 nM) to the extracellular domain ofthe human epidermal growth factor receptor2 protein, HER2 (ErbB2) (U.S.Pat. Nos. 5,821,337; 6,054,297; 6,407,213; 6,639,055; Coussens L, et al(1985) Science 230:1132-9; Slamon D J, et al (1989) Science 244:707-12).Trastuzumab contains human framework regions with thecomplementarity-determining regions of a murine antibody (4D5) thatbinds to HER2. Trastuzumab binds to the HER2 antigen and thus inhibitsthe growth of cancerous cells. Trastuzumab has been shown, in both invitro assays and in animals, to inhibit the proliferation of human tumorcells that overexpress HER2 (Hudziak R M, et al (1989) Mol Cell Biol9:1165-72; Lewis G D, et al (1993) Cancer Immunol Immunother; 37:255-63;Baselga J, et al (1998) Cancer Res. 58:2825-2831). Trastuzumab is amediator of antibody-dependent cellular cytotoxicity, ADCC (Hotaling TE, et al (1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res;37:471; Pegram M D, et al (1997) [abstract]. Proc Am Assoc Cancer Res;38:602; Sliwkowski et al (1999) Seminars in Oncology 26(4), Suppl12:60-70; Yarden Y. and Sliwkowski, M. (2001) Nature Reviews: MolecularCell Biology, Macmillan Magazines, Ltd., Vol. 2:127-137). HERCEPTIN® wasapproved in 1998 for the treatment of patients with ErbB2-overexpressingmetastatic breast cancers (Baselga et al, (1996) J. Clin. Oncol.14:737-744). The FDA approved HERCEPTIN® in 2006 as part of a treatmentregimen containing doxorubicin, cyclophosphamide and paclitaxel for theadjuvant treatment of patients with HER2-positive, node-positive breastcancer. There is a significant clinical need for developing furtherHER2-directed cancer therapies for those patients withHER2-overexpressing tumors or other diseases associated with HER2expression that do not respond, or respond poorly, to HERCEPTIN®treatment.

Pertuzumab (OMNITARG™, rhuMab 2C4, Genentech) is a clinical stage,humanized antibody and the first in a new class of agents known as HERdimerization inhibitors (HDIs) which block the ability of the HER2receptor to collaborate with other HER receptor family members, i.e.HER1/EGFR, HER3, and HER4 (U.S. Pat. No. 6,949,245; Agus et al (2002)Cancer Cell 2:127-37; Jackson et al (2004) Cancer Res 64:2601-9; Takaiet al (2005) Cancer 104:2701-8). In cancer cells, interfering withHER2's ability to collaborate with other HER family receptors blockscell signaling and may ultimately lead to cancer cell growth inhibitionand death of the cancer cell. HDIs, because of their unique mode ofaction, have the potential to work in a wide variety of tumors,including those that do not overexpress HER2 (Mullen et al (2007)Molecular Cancer Therapeutics 6:93-100).

Temozolomide, (CAS Reg. No. 85622-93-1, TEMODAR®, TEMODAL®, ScheringPlough) is a oral chemotherapy drug approved by the FDA for thetreatment of anaplastic astrocytoma, and has been studied for otherbrain tumor types such as glioblastoma multiforme (U.S. Pat. No.5,260,291; Stevens et al (1984) J. Med. Chem. 27:196; Newlands et al(1997) Cancer Treat. Rev. 23:35-61; Danson et al (2001) Expert Rev.Anticancer Ther. 1:13-19). Temozolomide is named as(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamideor 3,4-dihydro-3-methyl-4-oxoimidazo [5,1-d]-as-tetrazine-8-carboxamide(U.S. Pat. No. 5,260,291, CAS No. 85622-93-1), and has the structure:

Tamoxifen (NOLVADEX®, ISTUBAL®, VALODEX®) is an orally active, selectiveestrogen receptor modulator (SERM) which is used in the treatment ofbreast cancer and is currently the world's largest selling drug for thisindication. Tamoxifen (Nolvadex®) was first approved by the FDA (ICIPharmaceuticals, now AstraZeneca) in 1977 for treatment of metastaticbreast cancer (Jordan V C (2006) Br J Pharmacol 147 (Suppl 1): S269-76).Tamoxifen is currently used for the treatment of both early and advancedestrogen receptor (ER) positive breast cancer in pre- andpost-menopausal women (Jordan V C (1993) Br J Pharmacol 110 (2):507-17). It is also approved by the FDA for the prevention of breastcancer in women at high risk of developing the disease and for thereduction of contralateral (in the opposite breast) breast cancer.Tamoxifen is named as(Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine, (CASReg. No. 10540-29-1) and has the structure:

Doxorubicin (ADRIAMYCINID, hydroxyldaunorubicin) is a DNA-interactingdrug widely used in chemotherapy since the 1960s. It is an anthracyclineantibiotic and structurally related to daunomycin, which alsointercalates DNA. Doxorubicin is commonly used in the treatment of awide range of cancers. Doxorubicin is named as(8S,10S-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione,(CAS Reg. No. 23214-92-8) and has the structure:

Akti-1/2 is a cell-permeable quinoxaline compound that potently andselectively inhibits Akt1/Akt2 activity: IC₅₀=58 nM, 210 nM, and 2.12 μMfor Akt1, Akt2, and Akt3, respectively, in in vitro kinase assays(Barnett et al. (2005) Biochem. J. 385,:399; DeFeo-Jones, et al (2005)Mol. Cancer Ther. 4:271; Zhao et al (2005) Bioorg. Med. Chem. Lett.15:905; Lindsley et al (2005) Bioorg. Med. Chem. Lett. 15:761; US2006/142178; US 2005/159422; US 2004/102360). The inhibition appears tobe pleckstrin homology (PH) domain-dependent. It does not exhibit anyinhibitory effect against PH domain-lacking Akts, or other closelyrelated AGC family kinases, PKA, PKC, and SGK, even at concentrations ashigh as 50 μM. Akti-1/2 overcomes Akt1/Akt2-mediated resistance tochemotherapeutics in tumor cells and is shown to block basal andstimulated phosphorylation/activation of Akt1/Akt2 both in culturedcells in vitro and in mice in vivo. Akti-1/2 (EMD Biosciences ProductNo. 124018) is named as1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one,and has the structure:

HPPD is a selective B-Raf inhibitor (B-Raf IC50<2 nM, pERK IC50 87 nM)under preclinical investigations (US 2006/0189627). HPPD is named as5-(1-(2-hydroxyethyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-2,3-dihydro-1H-inden-1-oneoxime, and has the structure:

Rapamycin (sirolimus, RAPAMUNE®) is an immunosuppressant drug used toprevent rejection in organ transplantation, and is especially useful inkidney transplants. Rapamycin is a macrolide antibiotic (“-mycin”) firstdiscovered as a product of the bacterium Streptomyces hygroscopicus in asoil sample from an island called Rapa Nui, better known as EasterIsland (Pritchard D I (2005). Drug Discovery Today 10 (10): 688-691).Rapamycin inhibits the response to interleukin-2 (IL-2) and therebyblocks activation of T- and B-cells. The mode of action of rapamycin isto bind the cytosolic protein FK-binding protein 12 (FKBP12). Therapamycin-FKBP12 complex inhibits the mammalian target of rapamycin(mTOR) pathway through directly binding the mTOR Complex1 (mTORC1). mTORis also called FRAP (FKBP-rapamycin associated protein) or RAFT(rapamycin and FKBP target). Rapamycin is named as(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone(CAS Reg. No. 53123-88-9), and has the structure:

Lapatinib (TYKERB®, GW572016, Glaxo SmithKline) has been approved foruse in combination with capecitabine (XELODA®, Roche) for the treatmentof patients with advanced or metastatic breast cancer whose tumorsover-express HER2 (ErbB2) and who have received prior therapy includingan anthracycline, a taxane and trastuzumab. Lapatinib is anATP-competitive epidermal growth factor (EGFR) and HER2/neu (ErbB-2)dual tyrosine kinase inhibitor (U.S. Pat. Nos. 6,727,256; 6,713,485;7,109,333; 6,933,299; 7,084,147; 7,157,466; 7,141,576) which inhibitsreceptor autophosphorylation and activation by binding to theATP-binding pocket of the EGFR/HER2 protein kinase domain. Lapatinib isnamed asN-(3-chloro-4-(3-fluorobenzyloxy)phenyl)-6-(5-((2-(methylsulfonyl)ethylamino)methyl)furan-2-yl)quinazolin-4-amine,and has the structure:

Biological Evaluation

Certain Formula I and II compounds bind specifically to PI3 kinaseisoforms and inhibit the proliferation of tumor cells (WO 2006/046031;US 2008/0039459; US 2008/0076768; US 2008/0076758; WO 2008/070740; WO2008/073785).

Certain Formula Ia and IIa compounds bind the p110α isoform at IC50 lessthan 1 micromole and show single-agent in vivo tumor growth inhibitionin mouse xenograft models. Accordingly, Formula I and II compounds maybe used to treat a disease or disorder arising from abnormal cellgrowth, function or behavior as single agents or in combination therapywith one or more chemotherapeutic agents.

Certain exemplary Formula I and II compounds described herein wereprepared, characterized, and assayed for their PI3K binding activity(Example 13) and in vitro activity against tumor cells (Example 14). Therange of PI3K binding activities (IC50) was less than 1 nM (onenanomolar) to about 10 μM (ten micromolar). Certain exemplary Formula Iand II compounds have PI3K binding activity IC₅₀ values less than 10 nM.Certain Formula I and II compounds have tumor cell-based activity EC₅₀values less than 100 nM.

The cytotoxic or cytostatic activity of Formula I and II exemplarycompounds was measured by: establishing a proliferating mammalian tumorcell line in a cell culture medium, adding a Formula I or II compound,culturing the cells for a period from about 6 hours to about 5 days; andmeasuring cell viability (Example 14). Cell-based in vitro assays wereused to measure viability, i.e. proliferation (IC₅₀), cytotoxicity(EC₅₀), and induction of apoptosis (caspase activation). Pharmacodynamicand pharmacokinetic properties of absorption, distribution, metabolism,and excretion (ADME) were measured for certain exemplary compounds byassays including: Caco-2 Permeability, Hepatocyte Clearance, CytochromeP450 Inhibition, Cytochrome P450 Induction, Plasma Protein Binding, andhERG channel blockage.

In Vitro Cell Proliferation Assays

The in vitro potency of the combinations of Formula I or II compoundswith chemotherapeutic agents was measured by the cell proliferationassay of Example 14; the CellTiter-Glo® Luminescent Cell ViabilityAssay, commercially available from Promega Corp., Madison, Wis. Thishomogeneous assay method is based on the recombinant expression ofColeoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713; 5,700,670)and determines the number of viable cells in culture based onquantitation of the ATP present, an indicator of metabolically activecells (Crouch et al (1993) J. Immunol. Meth. 160:81-88; U.S. Pat. No.6,602,677). The CellTiter-Glo® Assay was conducted in 96 or 384 wellformat, making it amenable to automated high-throughput screening (HTS)(Cree et al (1995) AntiCancer Drugs 6:398-404). The homogeneous assayprocedure involves adding the single reagent (CellTiter-Glo® Reagent)directly to cells cultured in serum-supplemented medium. Cell washing,removal of medium and multiple pipetting steps are not required. Thesystem detects as few as 15 cells/well in a 384-well format in 10minutes after adding reagent and mixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CeliTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time.

The anti-proliferative effects of Formula I and II exemplary compoundsand combinations with chemotherapeutic agents were measured by theCellTiter-Glo® Assay (Example 14) against the tumor cell lines in FIGS.1-A, 1-B and 1-C. EC₅₀ values were established for the tested compoundsand combinations. The range of in vitro cell potency activities wasabout 100 nM to about 10 μM.

FIG. 1-A shows a summary of in vitro cell proliferation assays ofcombinations of Formula Ia compound and various chemotherapeutic agents.FIG. 1-B shows a summary of in vitro cell proliferation assays ofcombinations of Formula IIa compound and various chemotherapeuticagents. FIG. 1-C shows a summary of in vitro cell proliferation assaysof combinations of Formula Ib compound and various chemotherapeuticagents. The cancer cell lines are characterized by tumor type andpresence of gene mutation.

The individual measured EC50 values against the particular cell of theFormula Ia, Ib, and IIa compounds and of the chemotherapeutic agent arecompared to the combination EC50 value. The combination index (CI) scoreis calculated by the Chou and Talalay method (Chou, T. and Talalay, P.(1984) Adv. Enzyme Regul. 22:27-55). A CI less than 0.8 indicatessynergy. A CI between 0.8 and 1.2 indicates additivity. A CI greaterthan 1.2 indicates antagonism. The CI values in FIGS. 1-A, 1-B and 1-Care from the EC50 concentrations (third point from the right). Thestrength of synergy is assessed according to Chou and Talalay and listedin the last column of the tables. Certain combinations in FIGS. 1-A, 1-Band 1-C show the surprising and unexpected property of synergy in the invitro cell proliferation assays with tumor type cell lines includingbreast, cervical, colon, endometrial, glioma, lung, melanoma, ovarian,pancreatic, and prostate. Other combinations in FIGS. 1-A, 1-B and 1-Cshow no synergy; and only show mere additivity or antagonism. Certaincombinations are synergistic with one or more tumor types, but notothers. The synergy demonstrated in the in vitro cell proliferationassays provides a basis to expect a corresponding synergy in treatingcancers including, but not limited to, breast, cervical, colon,endometrial, glioma, lung, melanoma, ovarian, pancreatic, and prostatein human patients.

FIG. 2 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of 5-FU, Formula Ia compound, andthe combination of 5-FU and Formula Ia. The MDA-MB-361 (breast tumortype) cells are treated simultaneously (top), pre-dosed with Formula Ia4 hours before dosing with 5-FU (middle), and post-dosed with Formula Ia4 hours after dosing with 5-FU (bottom). Strong synergy is observed withsimultaneous dosing (CI=0.11) and post-dosing with Formula Ia (CI=0.10).A strong dosing order (sequence) effect is observed. Pre-dosing withFormula showed less synergy (CI=0.67).

FIG. 3 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of gemcitabine, Formula Ia compound,and the combination of gemcitabine and Formula Ia. The Cal-51 (breasttumor type) cells are treated simultaneously (top), and post-dosed withFormula Ia 4 hours after dosing with gemcitabine (bottom). Synergy isobserved with simultaneous dosing (CI=0.59) and strong synergy withpost-dosing with Formula Ia (CI=0.17).

FIG. 4 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of gemcitabine, Formula Ia compound,and the combination of gemcitabine and Formula Ia. The MDA-MB-361(breast tumor type) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with gemcitabine (middle), andpost-dosed with Formula Ia 4 hours after dosing with gemcitabine(bottom). Synergy is observed with simultaneous dosing (CI=0.27),pre-dosing with Formula Ia (CI=0.46), and post-dosing with Formula Ia(CI=0.28).

FIG. 5 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of erlotinib, Formula Ia compound,and the combination of erlotinib and Formula Ia. The A549 (lung tumortype, with K-ras G12C) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with erlotinib (middle), andpost-dosed with Formula Ia 4 hours after dosing with erlotinib (bottom).Synergy is observed with simultaneous dosing (CI=0.17), pre-dosing withFormula Ia (CI=0.31), and post-dosing with Formula Ia (CI=0.33).

FIG. 6 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of erlotinib, Formula Ia compound,and the combination of erlotinib and Formula Ia. The H23 (lung tumortype, with K-ras G12C mutation) cells are treated simultaneously (top),pre-dosed with Formula Ia 4 hours before dosing with erlotinib (middle),and post-dosed with Formula Ia 4 hours after dosing with erlotinib(bottom). Synergy is observed with simultaneous dosing (CI=0.28),pre-dosing with Formula Ia (CI=0.39), and post-dosing with Formula Ia(CI=0.37).

FIG. 7 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of temozolomide, Formula Iacompound, and the combination of temozolomide and Formula Ia. The U87(glioma tumor type) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with temozolomide (middle), andpost-dosed with Formula Ia 4 hours after dosing with temozolomide(bottom). Synergy is observed with simultaneous dosing (CI=0.004), butnot with pre-dosing with Formula Ia (CI=1.13), and post-dosing withFormula Ia (CI=1.41).

FIG. 8 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of temozolomide, Formula Iacompound, and the combination of temozolomide and Formula Ia. The A375(melanoma tumor type) cells are treated simultaneously (top), pre-dosedwith Formula Ia 4 hours before dosing with temozolomide (middle), andpost-dosed with Formula Ia 4 hours after dosing with temozolomide(bottom). Synergy is observed with simultaneous dosing (CI=0.007), butnot with pre-dosing with Formula Ia (CI=0.99), and post-dosing withFormula Ia (CI=1.02).

FIG. 9 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of temozolomide, Formula Iacompound, and the combination of temozolomide and Formula Ia. TheMALME-3M (melanoma tumor type) cells are treated simultaneously (top),pre-dosed with Formula Ia 4 hours before dosing with temozolomide(middle), and post-dosed with Formula Ia 4 hours after dosing withtemozolomide (bottom). Synergy is observed with simultaneous dosing(CI=0.18), but not with pre-dosing with Formula Ia (CI=1.46), andpost-dosing with Formula Ia (CI=1.22).

FIG. 10 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of doxorubicin, Formula Ia compound,and the combination of doxorubicin and Formula Ia. The SKOV3 (ovariantumor type) cells are treated simultaneously (top), pre-dosed withFormula Ia 4 hours before dosing with doxorubicin (middle), andpost-dosed with Formula Ia 4 hours after dosing with doxorubicin(bottom). Synergy is observed with simultaneous dosing (CI=0.39), andpost-dosing with Formula Ia (CI=0.18), but not with pre-dosing withFormula Ia (CI=1.44).

FIG. 11 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable cells over varying concentrations(starting at 4× EC50) right to left of docetaxel, Formula Ia compound,and the combination of docetaxel and Formula Ia. The PC3 (prostate tumortype) cells are treated simultaneously (top), pre-dosed with Formula Ia4 hours before dosing with docetaxel (middle), and post-dosed withFormula Ia 4 hours after dosing with docetaxel (bottom). Synergy isobserved with simultaneous dosing (CI=0.43), and post-dosing withFormula Ia (CI=0.30), but not with pre-dosing with Formula Ia (CI=1.23).

FIG. 12 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable MDA MD 361 (breast tumor type) cells overvarying concentrations (starting at 4× EC50) right to left of: (top)5-FU, Formula IIa compound, and the simultaneous combination of 5-FU andFormula IIa; (middle) docetaxel, Formula IIa compound, and thesimultaneous combination of docetaxel and Formula IIa; and (bottom)gemcitabine, Formula IIa compound, and the simultaneous combination ofgemcitabine and Formula IIa. Synergy is observed with simultaneousdosing of 5-FU and Formula IIa (CI=0.34), docetaxel and Formula IIa(CI=0.09), and gemcitabine and Formula IIa (CI=0.50).

FIG. 13 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring: (top) viable MT3 (breast tumor type) cells overvarying concentrations (starting at 4× EC50) right to left of docetaxel,Formula IIa compound, and the simultaneous combination of docetaxel andFormula IIa; and (bottom) viable U87 (glioma tumor type, PTEN negmutation) cells over varying concentrations (starting at 4× EC50) rightto left of temozolomide, Formula IIa compound, and the simultaneouscombination of temozolomide and Formula IIa. Synergy is observed withsimultaneous dosing in MT3 cells with docetaxel and Formula IIa(CI=0.69), and in U87 cells with temozolomide and Formula IIa (CI=0.67).

FIG. 14 shows results of in vitro cell proliferation assays (Cell-TiterGlo, Promega) measuring viable ZR75-1 (breast tumor type) cells overvarying concentrations (starting at 4× EC50) right to left of: (top)5-FU, Formula IIa compound, and the simultaneous combination of 5-FU andFormula IIa; and (bottom) docetaxel, Formula IIa compound, and thesimultaneous combination of docetaxel and Formula IIa. Synergy isobserved with simultaneous dosing in ZR75-1 cells with 5-FU and FormulaIIa (CI=0.47), and in ZR75-lcells with docetaxel and Formula IIa(CI=0.46).

The correlation between Ras mutations and the in vitro synergy effectslisted in FIG. 1A (Expts. 1-248) due to combinations of Formula Iacompound and chemotherapeutic agents can be displayed by dot plots. Eachdot in FIGS. 15 and 16 is an experiment from FIG. 1A. The experimentsare grouped as Ras wild-type (Ras WT) or Ras mutant (Ras Mut) with thespecific mutations noted in FIG. 1A, plotted against the synergy effect(Combination Index, CI), where synergy increases with decreasing CI ascalculated by the Chou and Talalay method (Chou, T. and Talalay, P.(1984) Adv. Enzyme Regul. 22:27-55).

FIG. 15 shows a dot plot of synergy (Combination Index) of erlotinib andFormula Ia compound experiments from FIG. 1A against tumor cell lineswith and without Ras mutations (Expts. 71-73, 140-168, 230-231). The rasmutant cell lines show stronger synergy between erlotinib and Formula Iacompound than ras wild-type cell lines.

FIG. 16 shows a dot plot of synergy (Combination Index) of PD-0325901and Formula Ia compound experiments from FIG. 1A against tumor celllines with and without Ras mutations (Expts. 29-35, 74-83, 124-139,175-184, 224-226, 232-236, 247, 248). The ras mutant cell lines showstronger synergy between PD-0325901 and Formula Ia compound than raswild-type cell lines.

FIG. 17 shows time-course results of treatment of a synergistic tumorcell line MDA-MB-361 and a non-synergistic tumor cell line MT-3 withgemcitabine at EC80 dosing levels. pAkt levels were measured at T=0(untreated, UT), 1 hr, 4 hr, 6 hr, and 24 hr. Constitutive and inducibleAkt activity is known to promote resistance to chemotherapy,trastuzumab, or tamoxifen in breast cancer cells (Clark et al (2002) MolCancer Ther. 1(9):707-17). Phospho Akt (pAkt) levels can be measured bythe method described in Example 18. Low CI correlates with the effect ofthe chemotherapeutic to induce an increase in pAkt. Levels of pAkt(Ser473) were determined using bead kits from Biosource (Carlsbad,Calif.) and the Luminex Bio-Plex system (Bio-Rad, Hercules, Calif.).Gemcitabine treatment leads to increased pAkt levels in the synergisticcell line (MDA-MB-361) but not in the non-synergistic cell line (MT-3),demonstrating that an increase in pAkt levels in response tochemotherapy is correlated and predictive of synergy by a Formula I orII compound and a chemotherapeutic agent in treatment of cancer.

FIG. 18 shows a dot plot of synergy (Combination Index) of docetaxel,5-FU, or gemcitabine and Formula Ia compound experiments from FIG. 1Aagainst tumor cell lines that show a pAkt increase or no pAkt increasein response to the chemotherapeutic agent alone. Cell lines that show anincrease in pAkt after treatment with docetaxel, 5-FU, or gemcitabineshow stronger synergy with Formula Ia compound than cell lines without apAkt response.

The invention includes a method for determining compounds to be used incombination for the treatment of cancer comprising: a) administering atherapeutic combination of a compound having Formula I or II, and achemotherapeutic agent selected from erlotinib, docetaxel, 5-FU,gemcitabine, PD-0325901, cisplatin, carboplatin, paclitaxel,bevacizumab, trastuzumab, pertuzumab, temozolomide, tamoxifen,doxorubicin, Akti-1/2, HPPD, rapamycin, and lapatinib to an in vitrotumor cell line with a K-ras mutation, and b) measuring a synergistic ornon-synergistic effect.

Although other mechanisms of action may be operative in the combinationsexemplified in FIGS. 1A, 1B, 1C, and FIGS. 2-14, the results areconsistent with PI3K inhibitors exerting a G1 phase specific effect onthe inhibition of tumor cell proliferation, 5FU exerting an S phasespecific disruption of DNA synthesis, gemcitabine exerting an S phasespecific disruption of DNA synthesis, and docetaxel exerting an M phasespecific depolarization of microtubules.

Flow Cytometry Facs

Flow cytometry was conducted to measure the effects of combinationtherapy of Formula Ia compound and several chemotherapeutic agents onMB3 breast tumor and PC3 prostate tumor cells. The Annexin V/PI assaydetects early and late apoptotic events (Example 15). Cells that areAnnexin V positive are in the early stages of apoptosis and those thatare both Annexin V and PI positive are noted as “dead” on the bar graphcharts of FIG. 19. The remaining cells make up the “live” population.

FIG. 19 shows results of flow cytometry FACS (Fluorescence ActivatedCell Sorter): (top) MB361 cells are (left to right) untreated, treatedwith Formula Ia, 5FU, and first with 5FU then Formula Ia compound;(middle) PC3 cells are (left to right) untreated, treated with FormulaIa, docetaxel, simultaneous with Formula Ia compound and docetaxel,first with Formula Ia then with docetaxel, and first with docetaxel,then with Formula Ia; and (bottom) MB361 cells are (left to right)untreated, treated Formula Ia, gemcitabine, and first with gemcitabinethen with Formula Ia.

Formula Ia compound and a genotoxic, chemotherapeutic agent were dosedat EC80 for 24, 48, 72 hours. FACS (Fixed for cell cycle (PI), livecells for Annexin V and PI) Compounds were added either simultaneously,or separated by 4 hours, pre-dosing and post-dosing with Formula Iacompound (Example 19). A wash out effect was noted where maximum synergywas attained after one hour. The combinations remained synergistic withboth drugs washed out one hour after dosing. There are increases inearly and late apoptosis in all three combinations (FIG. 19) whenFormula Ia is combined with the chemotherapeutic drug. A low Chou andTalalay CI value suggests that a significant benefit in tumor inhibitionis likely when these drugs are combined in vivo.

Three Dimensional Combination Assay

In glandular tissues such as the breast, the epithelium interacts with aspecialized form of extracellular matrix, known as basement membrane.The extracellular matrix regulates normal mammary gland biology andpathogenesis. Adaptation of a reconstituted, laminin-rich basementmembrane (1rBM) to standard cell culture can recapitulate the basicacinar architecture of the mammary gland and is considered to be animproved in vitro model to simulate the dynamic microenvironment of atumor (Debnath J, Brugge J S. (2005) Nat Rev Cancer. 5:675-88). Usingbroad specificity inhibitors, PI3K signaling has been implicated inacini development of HMT-3522 T4-2 human breast tumor cells grown in1rBM, whereby PI3K inhibition was sufficient to restore apical-basalpolarity and induce growth arrest (Liu H, Radisky D C, Wang F, Bissell MJ. (2004) J Cell Biol.; 164:603-12). Given the proposed contribution ofPI3K to the initiation and development of breast cancer, 3D culturesystems provide a novel and comprehensive means of assessing theefficacy of small-molecule PI3K inhibitors such as Formula Ia compound.

The receptor tyrosine kinase HER2 (Neu/ErbB2) is amplified andover-expressed in approximately 20% of human breast cancer and plays acausal role in mammary carcinogenesis (Yarden Y, Sliwkowski M X. (2001)Nat Rev Mol Cell Biol. 2:127-37). That HER2 over-expression plays a rolein human breast cancer is demonstrated by the therapeutic efficacy ofthe anti-HER2 monoclonal antibody, trastuzumab (HERCEPTIN®, Genentech).In addition to homodimerization, HER2 can also function as a co-receptorfor other HER family members. The monoclonal antibody pertuzumab targetsthe HER2 dimerization arm (extracellular subdomain II) and disrupts therecruitment of HER2 into HER receptor-ligand complexes (Franklin et al(2004) Cancer Cell. 5:317-28). The efficacy of PI3K inhibition wasdetermined using Formula Ia and IIa compounds combined with inhibitionof HER family signaling using the therapeutic antibodies, trastuzumaband pertuzumab.

The invention includes a method for determining compounds to be used incombination for the treatment of cancer comprising: a) administering atherapeutic combination of claim 1 to HER2-amplified breast cancer cellsin laminin-rich, reconstituted basement membrane media, wherein thechemotherapeutic agent targets, binds to, or modulates a HER2 receptor,and b) measuring inhibition of cellular proliferation whereinnonmalignant and malignant mammary cells are discriminated by one ormore phenotypic difference selected from cell viability and acinarmorphogenesis.

Combinations of Formula I and II compounds and therapeutic antibodieswere evaluated in HER2-amplified BT474M1 cells (Example 16). Cells werecultured in a 3-dimensional (3D) laminin-rich, reconstituted basementmembrane to account for the role of extracellular matrix molecules inoncogene signaling and biology. The ability of 3D culture torecapitulate the oncogenic microenvironment connotes that it may be amore reliable predictor of in vivo efficacy (as compared totwo-dimensional (2D) cultures of cells on plastic) and can be useful forthe characterization of inhibitors and target genes. 3D culture was usedto assess HER family signaling and measure the synergistic efficacy ofinhibitors. Cell viability and acinar phenotypes (morphogenesis) wereused as markers of drug efficacy (Example 16).

HER2-amplified BT474M1 cells were used to detect HER family signalingusing novel 3D cell culture phenotypes, and measure synergistic efficacybased on acinar morphogenesis (Example 16). One embodiment of thelaminin-rich, reconstituted basement membrane media isEngelbreth-Holm-Swarm extracellular matrix extract, commerciallyavailable as BD Matrigel™ (BD Biosciences). Another exemplary embodimentof an extracellular matrix (ECM) for 3D culture is Madin-Darby caninekidney epithelial cells. An exemplary phenotypic difference is theacinar architecture of invasive (malignant) and non-invasive(non-malignant) cells.

Acinar morphogenesis can be scored as an additional assay for drugefficacy. Each inhibitor is titrated to examine pathway pharmacodynamic(PD) markers, cell viability and acinar phenotype. The lowest inhibitordrug concentration was established that effectively inhibits the target.The suitable concentration of a Formula I and II compound for 3D cultureassays was determined by administering increasing doses withconsideration for overall cell viability, pharmacodynamics ofcorresponding downstream pathway markers (such as phosphorylated AKT1),and changes in acinar phenotype. The lowest drug concentration thateffectively inhibited the target was chosen for the assays. Aconcentration of 250 nM Formula Ia and IIa compound was selected as thefinal working concentration for 3D culture assays.

Direct inhibitors of HER2 such as trastuzumab and pertuzumab have beenshown to interfere with downstream activation of several key effectorpathways, including the PI3K-AKT axis. Combined inhibition of PI3K andHER family signaling in HER2-amplified breast cancer cells may result inpotent tumor cell inhibition.

By immunoblot detection of phosphorylated AKT1 (pAkt), 250 nM Formula Iacompound was confirmed to potently inhibit PI3K downstream signaling(Example 16). Therapeutic antibodies were used at saturatingconcentrations of 20 μg/ml and 25 μg/ml for pertuzumab and trastuzumab,respectively. Combination of Formula Ia and trastuzumab resulted in anadditive suppression of the 3D growth of BT474M1 acini cultured inMatrigel devoid of HRG ligand. To detect ligand-dependent HER2-HER3heterodimer signaling, as has been suggested to be transforming inmultiple cell lines, 1 nM HRG was added in these assays. Formula Ia andtrastuzumab had no effect on HRG-induced proliferation. By comparison,co-treatment with Formula Ia and pertuzumab resulted in an additivereduction of HRG-induced acini growth and morphogenesis. This effect wasshown to be statistically significant upon multiple replicates. In theabsence of ligand the reduction of 3D acini growth was greater followingtreatment with Formula Ia and trastuzumab as compared to Formula Ia andpertuzumab. The combination of trastuzumab and Formula Ia compoundinhibit cell proliferation and attenuate heregulin-inducedmorphogenesis. The combination of trastuzumab and Formula Ia compoundhave potent and additive effects on 3D growth in normal serum, but noadditivity was observed with the combination on acini growth ormorphogenesis in heregulin-treated media. The combination of pertuzumaband Formula Ia compound combination potently, but additively, inhibitsBT474M1 acini growth and morphogenesis. The triple combination oftrastuzumab, pertuzumab, and Formula Ia compound synergisticallyinhibits BT474M1 acini bud growth and morphogenesis (FIG. 20) in bothHRG-supplemented and standard media.

Each inhibitor was titrated to examine relevant pathway pharmacodynamic(PD) markers, cell viability, and acinar phenotype. The lowest inhibitordrug concentration that effectively inhibited the target was establishedand utilized in all 3D assays.

FIG. 20 shows the quantification of BT474 growth in three-dimensional(3D) culture. Cell viability was determined by measuring cellular ATPlevels. BT474M1 cells were cultured in either 10% serum or 10% serumwith 1 nM heregulin and subject to inhibitor combinations as indicated(left to right): media, DMSO, combination of 20 μg/ml trastuzumab and 25μg/ml pertuzumab, 250 nM Formula Ia compound, and the combination of 20μg/ml trastuzumab, 25 μg/ml pertuzumab, and 250 nM Formula Ia compound.Acini growth and morphogenesis is correlated with cellular ATPproduction in relative light units (RLU) in 10% FBS medium with andwithout 1 nM heregulin.

In standard media (without heregulin), cell activity is comparativelylower in the presence of trastuzumab, pertuzumab or Formula Ia compoundindividually, but not in HRG-treated media. The combination oftrastuzumab and Formula Ia compound inhibited cell proliferation andattenuated heregulin-induced morphogenesis in normal serum, but noadditivity was observed on acini growth or morphogenesis inheregulin-treated media. The combination of pertuzumab and Formula Iacompound potently, and additively, inhibited BT474M1 acini growth andmorphogenesis in both standard and heregulin-supplemented media. Thetriple combination of trastuzumab, pertuzumab, and Formula Ia compoundsynergistically inhibited BT474M1 cell proliferation andheregulin-induced morphogenesis (FIG. 20) in both standard andHRG-supplemented media. The combination of all three agentssynergistically decreases cell viability in both standard andHRG-supplemented media. Heregulin-induced morphogenesis of BT474M1 cellswas also abolished by the triple combination as determined throughmicroscopic inspection. These data suggest that the Formula Ia,trastuzumab and pertuzumab triple combination may provide improvedefficacy for treating HER2-amplified breast cancer in human patients.

Trastuzumab or Formula Ia significantly reduce acinar size in normalmedia, but have limited effect on HRG-induced morphogenesis. As acombined treatment, trastuzumab and Formula Ia can minimize acinar sizeand morphogenesis. By Cell Titer-Glo analysis of cell viability,additivity between trastuzumab and Formula Ia results in decreased cellgrowth in normal media, but no difference is seen with the addition ofHRG.

Pertuzumab completely inhibits HRG-induced morphogenesis, whereasFormula Ia only partially reduces the phenotype. Together, pertuzumaband Formula Ia abates cell growth and morphogenesis in both normal andHRG-supplemented media. By evaluation of cell viability as measured byCell Titer-Glo. a decrease in cellular activity is observed in thepresence of pertuzumab and Formula Ia as either single agents orcombined therapy in normal media. HRG-treated acini also show a similartrend, but to a lesser extent. By evaluation of Dunnett's T-testcomparison of Cell Titer-Glo replicates (n=8), the combination ofpertuzumab and Formula Ia significantly inhibits cellular activity(p=0.0054).

FIG. 21 shows a similar effect with the addition of Formula IIa to dualtrastuzumab and pertuzumab treatment. FIG. 21 shows BT474 growth in 3Dcell culture upon treatment of 20 μg/ml trastuzumab, 25 μg/ml pertuzumabor 250 nM Formula IIa compound, as indicated. Formula IIa significantlyreduced acinar size in normal media, but as a single agent had limitedeffect on HRG-induced morphogenesis. As a combined treatment, FormulaIIa, trastuzumab and pertuzumab significantly reduced acinar size andmorphogenesis, as determined by measurement of cell viability using CellTiter Glo. In comparison with Formula Ia compound, Formula IIa compoundis slightly less efficacious at 250 nM both as a single agent (p=0.0001,Dunnett's T-test) and in combination with trastuzumab and pertuzumab(p<0.0001, Dunnett's T-test).

FIG. 21-A shows a similar effect with the addition of Formula Ib to dualtrastuzumab and pertuzumab treatment. BT474 growth in 3D cell culturewas measured upon treatment of 20 μg/ml trastuzumab, 25 μg/ml pertuzumabor 20 nM Formula Ib compound, as indicated. Formula Ib monotherapyreduced acinar size in normal and HRG-supplemented media. The mostsignificant reduction in acinar size and morphogenesis, as determined bymeasurement of cell viability using Cell Titer Glo, resulted fromcombined treatment of Formula Ib, trastuzumab and pertuzumab.

In Vivo Tumor Xenograft Efficacy

The efficacy of the combinations of the invention may be measured invivo by implanting allografts or xenografts of cancer cells in rodentsand treating the tumor-bearing animals with the combinations. Variableresults are to be expected depending on the cell line, the presence orabsence of certain mutations in the tumor cells, the sequence ofadministration of Formula I or II compound and chemotherapeutic agent,dosing regimen, and other factors. Subject mice were treated withdrug(s) or control (Vehicle) and monitored over several weeks or more tomeasure the time to tumor doubling, log cell kill, and tumor inhibition(Example 17).

FIG. 22 shows the mean tumor volume change over time in CD-1 nude mice(Charles River Labs) with MDA-MB-361.1 breast tumor cell xenograftsdosed on day 0 with: MCT Vehicle (0.5% methylcellulose/0.2% Tween 80),150 mg/kg Formula Ia, 5 mg/kg docetaxel, and the combination of FormulaIa 150 mg/kg and docetaxel 5 mg/kg. Mice were dosed with docetaxel onday 1, 5 and 9 (q4d×3) intravenously while Formula 1a was dosed dailyfor 21 days by oral gavage. When administered on the same day, Formula1a was dosed 1 hour after docetaxel. The combination of 150 mg/kg offormula Ia with 5 mg/kg of docetaxel synergized to inhibit MDA-MB-361.1breast tumor growth in vivo greater than each single agent alone.

The group of 11 animals dosed with Formula Ia 150 mg/kg showed 75%inhibition after 21 days, and 3 partial regressions and 66% inhibitionafter 41 days. The group of 10 animals dosed with docetaxel 5 mg/kgshowed 78% inhibition after 21 days, and 2 partial regressions and 26%inhibition after 41 days. The group of 9 animals dosed with thecombination of Formula Ia 150 mg/kg and docetaxel 5 mg/kg showed 90%inhibition after 21 days, and 7 partial regressions and 83% inhibitionafter 41 days. The combination showed better efficacy in tumorinhibition and was statistically significant when compared to eachsingle drug (p=0.0001 vs docetaxel and p=0.02 vs Formula Ia).

FIG. 23 shows the mean tumor volume change over time in CD-1 nude mice(Charles River Labs) with MDA-MB-361.1 breast tumor cell xenograftsdosed on day 1 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80),37.5 mg/kg Formula IIa , 5 mg/kg docetaxel and the combination of 37.5mg/kg Formula IIa and 5 mg/kg. Mice were dosed with docetaxel on day 1,5 and 9 (q4d×3) intravenously while Formula IIa was dosed daily for 21days by oral gavage. When administered on the same day, Formula IIa wasdosed 1 hour after docetaxel. The group of 10 animals dosed with 37.5mg/kg Formula IIa showed 30% inhibition and 2 partial regressions after21 days. The group of 10 animals dosed with 5 mg/kg docetaxel showed 35%inhibition and 3 partial regressions after 21 days. The group of 10animals dosed with the combination of Formula IIa 37.5 mg/kg anddocetaxel 5 mg/kg showed 63% inhibition. The combination showed betterefficacy in tumor inhibition and was statistically significant whencompared to each single drug (p=0.0454 vs docetaxel and p=0.0174 vsFormula IIa).

FIG. 24 shows the mean tumor volume change over time in NMRI femalenu/nu (nude) mice with MAXF 401 (triple negative) primary breast tumorexplant xenografts dosed on day 0 with: MCT Vehicle (0.5%methycellulose/0.2% Tween 80), 100 mg/kg Formula Ia, 15 mg/kg docetaxeland the combination of 100 mg/kg Formula Ia and 15 mg/kg. MAXF 401 areprimary breast tumors biopsied directly from a patient that responded todocetaxel and implanted subcutaneously into mice. This breast cancergroup is a subpopulation of “triple negative” patients which are HER2negative, ER (estrogen receptor) negative, and PR (progesteronereceptor) negative. The sensitivity to docetaxel is retained in mice exvivo. Mice were dosed intravenously with docetaxel on day 0 and day 11while Formula Ia was dosed on day 0-4, 11-17 and 21-28 by oral gavage.Animals were monitored for an additional 22 days for tumor growth afterthe last dose of Formula Ia (total number of days animals were on-studywas 50). When administered on the same day, Formula Ia was dosed 1 hourafter docetaxel. The group of 10 animals dosed with 100 mg/kg Formula Iashowed 49% inhibition at day 28. The group of 10 animals dosed with 15mg/kg docetaxel showed 95% inhibition at day 28. The group of 10 animalsdosed with the combination of Formula Ia 150 mg/kg and docetaxel 15mg/kg showed >90% inhibition after 28 days. At the end of the study (day50), animals dosed with docetaxel and Formula Ia alone had regrowth oftheir tumors and tumor inhibition decreased from 95% to 68% and 49% to10%, respectively. However, At the end of the study (day 50), thecombination of docetaxel and Formula Ia resulted in all ten animalshaving sustained tumor regression (>90% inhibition) of MAXF 401 primarybreast tumors and was statistically significant when compared to eachsingle drug (p=0.05 vs docetaxel and p<0.001 vs Formula Ia). Given thatMAXF 401 breast tumors are derived from a patient that responded totaxane therapy, the improved efficacy observed ex vivo suggests that thecombination of Formula Ia may have clinical benefits in breast cancerwhen combined with docetaxel.

FIG. 25 shows the mean tumor volume change over time in NMRI femalenu/nu nude mice with MAXF 401 primary breast tumor explant xenograftsdosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80),100 mg/kg Formula IIa, 15 mg/kg docetaxel and the combination of 100mg/kg Formula IIa and 15 mg/kg docetaxel. MAXF 401 are primary breasttumors biopsied directly from a patient that responded to docetaxel andimplanted subcutaneously into mice. The sensitivity to docetaxel isretained in mice ex vivo. Mice were dosed intravenously with docetaxelon day 0 and day 11 while Formula IIa was dosed on day 0-3, 11-17 and21-28 by oral gavage. Animals were monitored for an additional 22 daysfor tumor growth after the last dose of Formula IIa (total number ofdays animals were on-study was 50). When administered on the same day,Formula IIa was dosed 1 hour after docetaxel. The group of 10 animalsdosed with 100 mg/kg Formula IIa showed 82% tumor inhibition at day 28.The group of 10 animals dosed with 15 mg/kg docetaxel showed 95% tumorinhibition at day 28. The group of 10 animals dosed with the combinationof Formula IIa 150 mg/kg and docetaxel 15 mg/kg showed 99% inhibition atday 28. At the end of the study (day 50), animals dosed with docetaxeland Formula IIa alone had regrowth of their tumors and tumor inhibitiondecreased to 68% and 51%, respectively. However, at the end of the study(day 50), the combination of 100 mg/kg Formula IIa and 15 mg/kgdocetaxel resulted in sustained tumor regression (99% inhibition) ofMAXF 401 primary breast tumors and was statistically significant whencompared to each single drug (p=0.0118 vs docetaxel and p=0.0005 vsFormula IIa). Given that MAXF 401 breast tumors are derived from apatient that responsed to taxane therapy, the improved efficacy observedex vivo suggests that the combination of formula IIa may have clinicalbenefits in breast cancer when combined with docetaxel.

FIG. 26 shows the mean tumor volume change over time in NMRI femalenu/nu nude mice with MAXF 1162 primary breast tumor explant xenograftsdosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80),100 mg/kg Formula Ia, 15 mg/kg docetaxel and the combination of 100mg/kg Formula Ia and 15 mg/kg docetaxel. MAXF 1162 are primary breasttumors biopsied directly from a patient that failed docetaxel therapyand implanted subcutaneously into mice. The resistance to docetaxel isretained ex vivo in these mice. Mice were dosed intravenously withdocetaxel on day 0, 11, 22 and 44 and Formula 1a was dosed on day 0-5,11-16, 22-27, 30-32, 42 and 44 by oral gavage. Animals were monitoredfor an additional 6 days for tumor growth after the last dose of FormulaIa (total number of days animals were on-study was 50). Whenadministered on the same day, Formula 1a was dosed 1 hour afterdocetaxel. The group of 10 animals dosed with 100 mg/kg Formula Iashowed 54% inhibition after 49 days. The group of 10 animals dosed with15 mg/kg docetaxel showed 36% inhibition after 49 days. The group of 10animals dosed with the combination of Formula Ia 100 mg/kg and docetaxel15 mg/kg showed 87% inhibition after 49 days. At the end of the study(day 50), the combination resulted in sustained tumor regression (>87%inhibition) and was statistically significant when compared to eachsingle drug (p=0.0005 vs docetaxel and p=0.0007 vs Formula Ia). Giventhat MAXF 1162 breast tumors are derived from a patient that failedtaxane therapy, the improved efficacy observed ex vivo suggests that thecombination of formula Ia with docetaxel may have clinical benefits intaxane-resistant breast cancer in humans.

FIG. 27 shows the mean tumor volume change over time in NMRI femalenu/nu nude mice with MAXF 1162 primary breast tumor xenografts dosed onday 0 with: MCT Vehicle (0.5% methycellulose/0.2% Tween 80), 100 mg/kgFormula IIa, 15 mg/kg docetaxel and the combination of 100 mg/kg FormulaIIa and 15 mg/kg docetaxel. MAXF 1162 are primary breast tumors biopsieddirectly from a patient that failed docetaxel therapy and implantedsubcutaneously into mice. The resistance to docetaxel is retained exvivo in these mice. Mice were dosed intravenously with docetaxel on day0, 11, 22 and 44 and Formula IIa was dosed on day 0-5, 11-16, 22-23,29-31 and 35-38 by oral gavage. Animals were monitored for an additional12 days for tumor growth after the last dose of Formula IIa (totalnumber of days animals were on-study was 50). When administered on thesame day, Formula IIa was dosed 1 hour after docetaxel. The group of 10animals dosed with 100 mg/kg Formula IIa showed 32% inhibition after 49days. The group of 10 animals dosed with 15 mg/kg docetaxel showed 36%inhibition after 49 days. The group of 10 animals dosed with thecombination of Formula IIa 100 mg/kg and docetaxel 15 mg/kg showed 80%inhibition after 49 days. At the end of the study (day 50), thecombination resulted in sustained tumor regression (>80% inhibition) ofMAXF 1162 primary breast cancer tumors and was statistically significantwhen compared to each single drug (p<0.0001 vs docetaxel and p=0.0166 vsFormula IIa). Given that MAXF 1162 breast tumors are derived from apatient that failed taxane therapy, the improved efficacy observed exvivo suggests that the combination of formula IIa with docetaxel mayhave clinical benefits in taxane-resistant human breast cancer.

FIG. 28 shows the mean tumor volume change over time in CRL female nu/nu(nude) mice with NCI-H2122 non-small cell lung cancer (NSCLC) tumorxenografts dosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2%Tween 80), 50 mg/kg Formula Ia, 75 mg/kg erlotinib and the combinationof 50 mg/kg Formula Ia and 75 mg/kg erlotinib. Mice were dosed dailywith erlotinib and Formula Ia daily for 16 days by oral gavage. Animalswere monitored for tumor growth for an additional 5 days (end of studywas day 21). Both erlotinib and Formula Ia were administeredsimultaneously. The group of 8 animals dosed with 50 mg/kg Formula Iashowed 17% tumor inhibition after 20 days. The group of 8 animals dosedwith 75 mg/kg erlotinib showed 21% inhibition after 20 days. The groupof 8 animals dosed with the combination of Formula Ia 50 mg/kg anderlotinib 75 mg/kg showed 55% inhibition after 20 days. At the end ofthe study (day 21), the combination of 50 mg/kg Formula Ia and erlotinibwas additive and resulted in significant tumor growth delay of NCI-H2122NSCLC tumor xenografts when compared to each single drug (p=0.032 vserlotinib and p=0.019 vs Formula Ia).

FIG. 29 shows the mean tumor volume change over time in CRL female nu/nu(nude) mice with NCI-H2122 non-small cell lung cancer (NSCLC) tumorxenografts dosed on day 0 with: MCT Vehicle (0.5% methycellulose/0.2%Tween 80), 50 mg/kg Formula IIa, 75 mg/kg erlotinib and the combinationof 50 mg/kg Formula IIa and 75 mg/kg erlotinib. Mice were dosed dailywith erlotinib and Formula IIa for 14 days (end of study) by oralgavage. Both erlotinib and Formula IIa were administered simultaneously.The group of 9 animals dosed with 50 mg/kg Formula IIa showed 27% tumorinhibition at the end of study. The group of 10 animals dosed with 75mg/kg erlotinib showed 34% tumor inhibition at the end of study. Thegroup of 9 animals dosed with the combination of Formula IIa 50 mg/kgand erlotinib 75 mg/kg showed 63% tumor inhibition at the end of study.At the end of the study (day 21), the combination of 50 mg/kg FormulaIIa and 75 mg/kg erlotinib was additive and resulted in significanttumor growth delay of NSCLC tumor xenografts when compared to eachsingle drug (p=0.032 vs erlotinib and p=0.029 vs Formula IIa).

FIG. 30 shows the mean tumor volume change over time in HRLN femalenu/nu mice with MCF-7 (PI3K mutant) breast tumor cell xenografts dosedon day 1 with: MCT and PBS vehicle (MCT; 0.5% methycellulose/0.2% Tween80 and PBS; phosphate buffered saline), control IgG 5 mg/kg, mB20-4.1murine anti-VEGF (anti-angiogenic) 5 mg/kg, Formula Ia 150 mg/kg, andthe combination of Formula Ia 150 mg/kg and mB20-4.1 murine anti-VEGF 5mg/kg. Animals were dosed with control IgG and mB20-4.1 intraperitonealytwice a week for 3 weeks and with Formula Ia daily for 21 days by oralgavage and tumor growth was monitored for an additional 41 days (totalnumber of day on-study was 62). Formula Ia and mB20-4.1 wereco-administered simultaneously. The group of 13 out of 15 animals dosedand on-study with control IgG showed 19% inhibition and 0 partialregressions after 21 days. The group of 10 out of 15 animals dosed andon-study with mB20-4.1 murine anti-VEGF showed 49% inhibition and 0partial regressions after 21 days. The group of 13 animals dosed andon-study with 150 mg/kg Formula Ia compound showed 36% inhibition and 0paritial regressions after 21 days. The group of 10 animals dosed andon-study with the combination of Formula Ia compound and mB20-4.1 murineanti-VEGF showed 66% inhibition and 4 complete regresssions after 21days. Animals dosed with the combination of Formula Ia and mB20-4.1murine anti-VEGF showed a significant inhibition and sustainedregression of tumor growth 41 days after dosing was terminated (end ofstudy) when compared to each single drug (p<0.006 vs Formula 1a and<0.01 vs mB20-4.1).

FIG. 31 shows the mean tumor volume change over time in HRLN femalenu/nu mice with MCF-7 (PI3K mutant) breast tumor cell xenografts dosedon day 1 with: MCT and PBS vehicle (MCT; 0.5% methycellulose/0.2% Tween80 and PBS; phosphate buffered saline), control IgG 5 mg/kg, mB20-4.1murine anti-VEGF (anti-angiogenic) 5 mg/kg, Formula IIa 100 mg/kg, andthe combination of Formula IIa 100 mg/kg and mB20-4.1 murine anti-VEGF 5mg/kg. Animals were dosed with control IgG and mB20-4.1 intraperitonealytwice a week for 3 weeks and with Formula IIa orally daily for 21 daysand tumor growth was monitored for an additional 41 days (total numberof day on-study was 62). Formula IIa and mB20-4.1 were co-administeredsimultaneously. The group of 12 out of 15 animals dosed and on-studywith vehicle showed 10% inhibition and 0 partial regressions after 21days. The group of 10 out of 15 animals dosed and on-study with mB20-4.1murine anti-VEGF showed 49% inhibition and 0 partial regressions after21 days. The group of 13 animals dosed and on-study with 100 mg/kgFormula IIa compound showed 5% inhibition and 0 partial regressionsafter 21 days. The group of 10 out of 15 animals dosed and on-study withthe combination of Formula IIa compound and mB20-4.1 murine anti-VEGFshowed 61% inhibition and 1 complete regression after 21 days . Animalsdosed with the combination of Formula Ia and mB20-4.1 murine anti-VEGFshowed a significant inhibition and delay in tumor growth 41 days afterdosing was terminated when compared to each single drug (p<0.001 vsFormula IIa and <0.01 vs mB20-4.1).

FIG. 32 shows the mean tumor volume change over time with Harlan femalenu/nu mice with U87MG glioma tumor cell xenografts dosed on day 0 with:Formula Ia (GDC-0941) 109 mg/kg, temozolomide 100 mg/kg, and thecombination of Formula Ia 109 mg/kg and temozolomide 100 mg/kg, alongwith mice receiving no drug (No Treatment group). Animals were dosedwith Formula Ia orally daily for 21 days, and temozolomide orally dailyfor 5 days. The combination of 109 mg/kg of Formula Ia with 100 mg/kg oftemozolomide synergized to inhibit U87MG glioma tumor growth in vivogreater than Formula Ia or temozolomide alone and resulted in tumorregression followed by tumor growth delay.

FIG. 33 shows the mean tumor volume change over time with CD-1 nudeCR/Hollister mice with MDA-MB-361.1 breast tumor cell xenografts dosedon day 0 with: Formula Ia (GDC-0941) 150 mg/kg, gemcitabine 100 mg/kg,and the combination of Formula Ia 150 mg/kg and gemcitabine 100 mg/kg,along with mice receiving no drug (Vehicle group). Animals were dosedwith Formula Ia orally daily for 21 days, and gemcitabineintraperitonealy on days 1, 4, 7 and 10 (q3d×4). The combination of 150mg/kg of Formula Ia with 100 mg/kg of gemcitabine synergized to inhibitMDA-MB-361.1 breast tumor growth in vivo greater than Formula Ia orgemcitabine alone and resulted in tumor regression followed by tumorgrowth delay.

FIG. 34 shows the mean tumor volume change over time with Harlan femalenu/nu mice with BT474 breast tumor cell xenografts dosed on day 0 with:Formula Ia (GDC-0941) at 18, 36, and 73 mg/kg, trastuzumab 20 mg/kg, andthe combinations of Formula Ia at 18, 36, and 73 mg/kg and trastuzumab20 mg/kg, along with mice receiving no drug (Vehicle group). Animalswere dosed with Formula Ia orally daily for 21 days, and trastuzumabintraperitonealy twice a week for 3 weeks. The combination of 73 mg/kgof Formula Ia with 20 mg/kg of trastuzumab synergized to inhibit BT474breast tumor growth in vivo greater than Formula Ia or trastuzumab aloneand resulted in tumor regression followed by tumor growth delay.

Treatment of BT474-M1 (in vivo passaged subclone of BT474) cells withtrastuzumab caused a decrease in pAKT equal to the decrease achievedwith Formula Ia compound (GDC-0941). Adding trastuzumab to 50 nM FormulaIa caused a dose dependent, enhanced, decrease in pAKT. At the maximaltrastuzumab dose, the additional reduction was 29-38% of GDC-0941treatment alone. The enhanced combinatorial effect on pAKT inhibitionwas not transient and could still be detected 48 h after treatment. Thecombinatorial effect seen in pAKT was also reflected in downstream AKTsignaling components. BT474-M1 cells were treated for four hours withFormula Ia in the presence or absence of trastuzumab. The addition oftrastuzumab further decreased the phosphorylation of direct AKTsubstrate PRAS40 (Thr246) and a distal substrate, phospho-S6 ribosomalprotein (Ser235/236), suggesting that trastuzumab and Formula Ia haveenhanced combinatorial effect on downstream AKT signaling. EnhancedPI3K/AKT pathway inhibition results in decreased cellproliferation/viability was demonstrated when BT474-M1 cells weretreated for six days and cell viability was measured. Trastuzumab alonereduced proliferation/viability by 40%. In the absence of trastuzumab,the IC₅₀ value for Formula Ia was 296 nM. The addition of trastuzumabcaused a dose-dependent, enhanced reduction in proliferation/viability.A maximal trastuzumab dose of 10 ug/ml resulted in a 64% decrease in theconcentration of Formula Ia that is required to reach its IC50 of 106nM, demonstrating an enhanced combinatorial effect in decreasing cellproliferation/viability as well. A similar combinatorial effect on pAKTand inhibition of proliferation was also observed when SKBR-3 cells weretreated, but not when the trastuzumab non-responsive KPL-4 cells weretreated. The combination of trastuzumab and Formula Ia inhibitsproliferation synergistically in BT474 and SKBR-3 cells over most of theeffective drug range as determined by CalcuSyn software, showing thatthe combination enhances the inhibitory effect on AKT and its downstreamtargets resulting in a synergistic effect on the proliferation oftrastuzumab sensitive breast cancer cells.

The combination of trastuzumab and Formula Ia additively inducesapoptosis of BT474-M1 cells breast cancer cells treated for 48 h. Thecombination of trastuzumab and Formula Ia increased the accumulation ofcleaved caspase-3 fragments, indicative of activation of this keyeffector caspase. Combining trastuzumab and Formula Ia also resulted inan increase in cleaved PARP 89 kDa fragment, a known response tocaspase-3 activation. The activity of caspases 3 and 7 was alsoincreased when trastuzumab was added to Formula Ia treatment. Combiningtrastuzumab with 250 nM Formula Ia increased the activity of caspases 3and 7 to a level similar to that detected with a four fold higher doseof (1000 nM) Formula Ia alone. Importantly, this increase in caspaseactivity was reflected on the apoptotic index of these cells. Theaddition of trastuzumab dramatically decreased the concentration ofFormula Ia required to induce apoptosis. A near equivalent level ofapoptosis was detected when cells were treated with 100 nM of Formula Iaand trastuzumab than when treated with 1000 nM of Formula Ia alone. Asexpected, the increase in apoptosis was reflected in a decrease in cellviability after 48 h. A similar increase in caspase activity andapoptosis was seen when SKBR-3 cells were treated with the inhibitorcombination. The combination of Formula Ia with trastuzumabsignificantly lowers the Formula Ia concentration required for reachingthe threshold of caspase activation and apoptosis in trastuzumabsensitive breast cancer cells. Therefore, trastuzumab treatment could beused to sensitize HER2 amplified cells to PI3K inhibition and thusprovide an additional level of tumor specificity for the PI3K inhibitorFormula Ia.

FIG. 35 shows the mean tumor volume change over time with Harlan femalenu/nu mice with BT474 breast tumor cell xenografts dosed on day 0 with:Formula Ib at 2.5 mg/kg orally daily for 3 weeks, Formula Ib at 2.5mg/kg orally twice a week for 3 weeks, Formula Ib at 5 mg/kg orallydaily for 3 weeks, trastuzumab 15 mg/kg intraperitonealy once a week for3 weeks, and the combinations of: Formula Ib at 2.5 mg/kg orally dailyfor 3 weeks and trastuzumab 15 mg/kg intraperitonealy once a week for 3weeks; Formula Ib at 2.5 mg/kg orally twice a week for 3 weeks andtrastuzumab 15 mg/kg intraperitonealy once a week for 3 weeks; andFormula Ib at 5 mg/kg orally daily for 3 weeks and trastuzumab 15 mg/kgintraperitonealy once a week for 3 weeks, along with mice receiving nodrug (Vehicle group). The combination of 2.5 mg/kg of Formula Ib doseddaily with 15 mg/kg of trastuzumab dosed once a week synergized toinhibit BT474 breast tumor growth in vivo greater than Formula Ia ortrastuzumab alone and resulted in tumor growth delay.

FIG. 36 shows the mean tumor volume change over time with Harlan femalenu/nu mice with MCF-7 breast tumor cell xenografts dosed on day 0 with:murine anti-VEGF antibody B20-4.1 5 mg/kg intraperitonealy twice a weekfor 3 weeks, or Formula Ib at 5 mg/kg and the combinations of: FormulaIb at 5 mg/kg orally daily for days 0-3, 10-26 and B20-4.1 5 mg/kgintraperitonealy twice a week for 3 weeks along with mice receiving nodrug (Vehicle group). The combination of 5.0 mg/kg of Formula Ib with 5mg/kg B20-4.1 synergized to inhibit MCF-7 breast tumor growth in vivogreater than Formula Ib or B20-4.1 alone and resulted in tumorregression.

FIG. 37 shows the mean tumor volume change over time with Harlan femalenu/nu mice with Fo5 breast tumor cell xenografts dosed on day 0 with:murine anti-VEGF antibody B20-4.1 5 mg/kg intraperitonealy twice a weekfor 3 weeks, Formula Ia (GDC-0941) at 36 and 73 mg/kg orally daily for21 days, Formula Ib at 2.5 and 5 mg/kg orally daily for 21 days, and thecombinations of: Formula Ia at 36 mg/kg orally daily for 21 days andB20-4.1 5 mg/kg intraperitonealy twice a week for 3 weeks; Formula Ia at73 mg/kg orally daily for 21 days and B20-4.1 5 mg/kg intraperitonealytwice a week for 3 weeks; Formula Ib at 5 mg/kg orally daily for 21 daysand B20-4.1 5 mg/kg intraperitonealy twice a week for 3 weeks, andFormula Ib at 2.5 mg/kg orally daily for 21 days and B20-4.1 5 mg/kgintraperitonealy twice a week for 3 weeks along with mice receiving nodrug (Vehicle group). The combination of 36 mg/kg of Formula Ia with 5mg/kg B20-4.1 synergized to inhibit Fo5 breast tumor growth in vivogreater than Formula Ia or B20-4.1 alone and resulted in tumor growthdelay. The combination of 73 mg/kg of Formula Ia with 5 mg/kg B20-4.1also synergized to inhibit Fo5 breast tumor growth in vivo greater thanFormula Ia or B20-4.1 alone and resulted in tumor growth delay. Thecombination of 2.5 mg/kg of Formula Ib with 5 mg/kg B20-4.1 synergizedto inhibit Fo5 breast tumor growth in vivo greater than Formula Ib orB20-4.1 alone and resulted in tumor growth delay. The combination of 5.0mg/kg of Formula Ib with 5.0 mg/kg B20-4.1 synergized to inhibit Fo5breast tumor growth in vivo greater than Formula Ib or B20-4.1 alone andresulted in tumor growth regression.

FIG. 38 shows the mean tumor volume change over time with Harlan femalenu/nu mice with MDA-MB-231 breast tumor cell xenografts dosed on day 0with: murine anti-VEGF antibody B20-4.1 5 mg/kg intraperitonealy twice aweek for 3 weeks, Formula Ia (GDC-0941) at 36 and 73 mg/kg orally dailyfor 21 days, Formula Ib at 5 mg/kg orally daily for 21 days and FormulaIb at 7.5 mg/kg orally daily for 8 days, and the combinations of:Formula Ia at 36 mg/kg orally daily for 21 days and B20-4.1 5 mg/kgintraperitonealy twice a week for 3 weeks; Formula Ia at 73 mg/kg orallydaily for 21 days and B20-4.1 5 mg/kg intraperitonealy twice a week for3 weeks; Formula Ib at 5 mg/kg orally daily for 21 days and B20-4.1 5mg/kg intraperitonealy twice a week for 3 weeks; and Formula Ib at 7.5mg/kg orally daily for 8 days and B20-4.1 5 mg/kg intraperitonealy twicea week for 1.5 weeks, along with mice receiving no drug (Vehicle group).The combination of 5.0 mg/kg of Formula Ib with 5.0 mg/kg B20-4.1synergized to inhibit MDA-MB-231 breast tumor growth in vivo greaterthan Formula Ib or B20-4.1 alone and resulted in tumor growth delay.

FIG. 39 shows the mean tumor volume change over time with Harlan femalenu/nu mice with H1299 non-small cell lung cancer (NSCLC) tumor cellxenografts dosed on day 0 with: erlotinib 50 mg/kg orally daily for 3weeks, Formula Ia (GDC-0941) at 100 mg/kg orally daily for 6 days,Formula Ia at 50 mg/kg orally daily for 21 days, Formula Ia 25 mg/kgorally daily for 21 days, and the combinations of Formula Ia 100 mg/kgorally daily for 6 days and erlotinib 50 mg/kg orally daily for 6 days;Formula Ia 50 mg/kg orally daily for 21 days and erlotinib 50 mg/kgorally daily for 21 days; and Formula Ia 25 mg/kg orally daily for 21days and erlotinib 50 mg/kg orally daily for 21 days, along with micereceiving no drug (Vehicle group). The combination of 25 mg/kg ofFormula Ia with 50 mg/kg of erlotinib synergized to inhibit H1299non-small cell lung cancer tumor growth in vivo greater than Formula Iaor erlotinib alone and resulted in tumor growth delay. The combinationof 50 mg/kg of Formula Ia with 50 mg/kg of erlotinib also synergized toinhibit NCI-H1299 non-small cell lung cancer tumor growth in vivogreater than Formula Ia or erlotinib alone and resulted in tumor growthdelay.

FIG. 40 shows the mean tumor volume change over time with Harlan femalenu/nu mice with H520 non-small cell lung cancer (NSCLC) tumor cellxenografts dosed on day 0 with: erlotinib 50 mg/kg orally daily for 3weeks, Formula Ia (GDC-0941) at 73 mg/kg orally daily for 4 days,Formula Ia at 36 mg/kg orally daily for 21 days, Formula Ia 18 mg/kgorally daily for 21 days, and the combinations of Formula Ia 73 mg/kgorally daily for 4 days and erlotinib 50 mg/kg orally daily for 4 days;Formula Ia 36 mg/kg orally daily for 21 days and erlotinib 50 mg/kgorally daily for 21 days; and Formula Ia 18 mg/kg orally daily for 21days and erlotinib 50 mg/kg orally daily for 21 days, along with micereceiving no drug (Vehicle group). The combination of 18 mg/kg ofFormula Ia with 50 mg/kg of erlotinib synergized to inhibit H520non-small cell lung cancer tumor growth in vivo greater than Formula Iaor erlotinib alone and resulted in tumor growth delay. The combinationof 36 mg/kg of Formula Ia with 50 mg/kg of erlotinib also synergized toinhibit H520 non-small cell lung cancer tumor growth in vivo greaterthan Formula Ia or erlotinib alone and resulted in tumor growth delay.

FIG. 41 shows the mean tumor volume change over time with Harlan femalenu/nu mice with H1299 non-small cell lung cancer (NSCLC) tumor cellxenografts dosed on day 0 with: erlotinib 50 mg/kg orally daily for 21days, Formula Ib at 2.5 mg/kg orally daily for 21 days, Formula Ib at 5mg/kg orally twice per week for 21 days, Formula Ib at 5 mg/kg orallyonce per week for 3 weeks, and the combinations of: Formula Ib at 2.5mg/kg orally daily for 21 days and erlotinib 50 mg/kg orally daily for21 days; Formula Ib at 5 mg/kg orally twice per week for 3 weeks anderlotinib 50 mg/kg orally daily for 21 days; and Formula Ib at 5 mg/kgorally once per week for 3 weeks and erlotinib 50 mg/kg orally daily for21 days, along with mice receiving no drug (Vehicle group). Thecombination of 2.5 mg/kg of Formula Ib dosed orally daily with 50 mg/kgof erlotinib dosed orally daily for 21 days synergized to inhibit H520non-small cell lung cancer tumor growth in vivo greater than Formula Ibor erlotinib alone and resulted in tumor growth delay.

FIG. 42 shows the mean tumor volume change over time with Taconic NCRfemale nude mice with NCI-H2122 non-small cell lung cancer (NSCLC) tumorcell xenografts dosed on day 0 with: erlotinib 75 mg/kg orally daily for16 days, Formula Ib at 2.5 mg/kg orally daily for 16 days, Formula Ib at5 mg/kg orally daily for 16 days, Formula Ib at 7.5 mg/kg orally dailyfor 16 days, and the combinations of: Formula Ib at 2.5 mg/kg orallydaily for 16 days and erlotinib 50 mg/kg orally daily for 16 days;Formula Ib at 5 mg/kg orally daily for 16 days and erlotinib 50 mg/kgorally daily for 16 days; and Formula Ib at 7.5 mg/kg orally daily for16 days and erlotinib 50 mg/kg orally daily for 16 days, along with micereceiving no drug (Vehicle group). The combination of 2.5 mg/kg ofFormula Ib with 75 mg/kg of erlotinib synergized to inhibit NCI-H2122non-small cell lung cancer tumor growth in vivo greater than Formula Ibor erlotinib alone and resulted in tumor stasis.

FIG. 43 shows the mean tumor volume change over time with Harlan femalenu/nu mice with A375 human melanoma cancer cell xenografts dosed on day0 with: PD-0325901 3 mg/kg orally daily for 3 weeks, Formula Ia(GDC-0941) at 73 mg/kg orally daily for 3 weeks, and the combination of:PD-0325901 3 mg/kg orally daily for 3 weeks and Formula Ia 73 mg/kgorally daily for 3 weeks, along with mice receiving no drug (Vehiclegroup). The combination of 73 mg/kg of Formula Ia with 3 mg/kg ofPD-0325901 synergized to inhibit A375 human melanoma tumor growth invivo greater than Formula Ia or PD-0325901 alone and resulted in tumorregression and tumor growth delay.

FIG. 44 shows the mean tumor volume change over time with Harlan femalenu/nu mice with A375 human melanoma cancer tumor cell xenografts dosedon day 0 with: temozolomide 100 mg/kg orally daily for 5 days, FormulaIb at 10 mg/kg orally once weekly for 3 weeks, Formula Ib at 5 mg/kgorally weekly for 3 weeks, and the combinations of: Formula Ib at 10mg/kg orally once weekly for 3 weeks and temozolomide 100 mg/kg orallydaily for 5 days; and Formula Ib at 5 mg/kg orally once weekly for 3weeks and temozolomide 100 mg/kg orally daily for 5 days, along withmice receiving no drug (Vehicle group). The combination of 5 mg/kg ofFormula Ib with 100 mg/kg of temozolomide synergized to inhibit A375human melanoma tumor growth in vivo greater than Formula Ib ortemozolomide alone and resulted in in tumor growth delay.

FIG. 45 shows the mean tumor volume change over time with Harlan femalenu/nu mice with SKOV3 human ovarian cancer cell xenografts dosed on day0 with: Formula Ia (GDC-0941) 73 mg/kg orally daily for 3 weeks, FormulaIa 36 mg/kg orally daily for 3 weeks, docetaxel 10 mg/kg intravenouslyweekly for 3 weeks, and the combinations of: Formula Ia 73 mg/kg orallydaily for 3 weeks and docetaxel 10 mg/kg intravenously weekly for 3weeks; Formula Ia 36 mg/kg orally daily for 3 weeks and docetaxel 10mg/kg intravenously weekly for 3 weeks; and Formula Ia 73 mg/kg orallyweekly for 3 weeks and docetaxel 10 mg/kg intravenously weekly for 3weeks, along with mice receiving no drug (Vehicle group). Thecombination of 36 mg/kg dosed daily of Formula Ia with 10 mg/kg ofdocetaxel synergized to inhibit SKOV3 human ovarian tumor growth in vivogreater than Formula Ib or docetaxel alone and resulted in tumor growthdelay. The combination of 73 mg/kg of Formula Ia dosed daily with 10mg/kg of docetaxel also synergized to inhibit SKOV3 human ovarian tumorgrowth in vivo greater than Formula Ib or docetaxel alone and resultedin tumor growth delay.

FIG. 46 shows the mean tumor volume change over time with Harlan femalenu/nu mice with SKOV3 human ovarian cancer tumor cell xenografts dosedon day 0 with: Formula Ib 5 mg/kg orally daily for 3 weeks, Formula Ib 1mg/kg orally daily for 3 weeks, docetaxel 10 mg/kg intravenously weeklyfor 3 weeks, and the combinations of: Formula Ib 5 mg/kg orally dailyfor 3 weeks and docetaxel 10 mg/kg intravenously weekly for 3 weeks; andFormula Ib 1 mg/kg orally daily for 3 weeks and docetaxel 10 mg/kgintravenoulsy weekly for 3 weeks; along with mice receiving no drug(Vehicle group). The combination of 5 mg/kg of Formula Ib with 10 mg/kgof docetaxel synergized to inhibit SKOV3 human ovarian tumor growth invivo greater than Formula Ib or docetaxel alone and resulted in tumorstasis.

FIG. 47 shows the mean tumor volume change over time with Harlan femalenu/nu mice with SKOV3 human ovarian cancer tumor cell xenografts dosedon day 0 with: Formula Ib 5 mg/kg orally weekly for 3 weeks, Formula Ib10 mg/kg orally weekly for 3 weeks, docetaxel 10 mg/kg intravenouslyweekly for 3 weeks, and the combinations of: Formula Ib 5 mg/kg orallyweekly for 3 weeks and docetaxel 10 mg/kg intravenously weekly for 3weeks; and Formula Ib 10 mg/kg orally weekly for 3 weeks and docetaxel10 mg/kg intravenously weekly for 3 weeks, along with mice receiving nodrug (Vehicle group). The combination of 10 mg/kg of Formula Ib dosedorally weekly with 10 mg/kg of docetaxel synergized to inhibit SKOV3human ovarian tumor growth in vivo greater than Formula Ib or docetaxelalone and resulted in tumor growth delay.

FIG. 48 shows the mean tumor volume change over time with female SCIDBeige nude mice with LuCap 35V human primary prostate cancer tumor cellxenografts dosed on day 0 with: docetaxel 5 mg/kg intravenously on days1, 5 and 9 (q4d×3) Formula Ia (GDC-0941) 50 mg/kg orally daily for 18days Formula Ia 100 mg/kg orally daily for 18 days and the combinationsof: docetaxel 5 mg/kg intravenously on days 1, 5 and 9 (q4d×3) andFormula Ia 50 mg/kg orally daily for 18 days and docetaxel 5 mg/kgintravenously on days 1, 5 and 9 (q4d×3) and Formula Ia 100 mg/kg orallydaily for 18 days, along with mice receiving no drug (Vehicle group).The combination of 100 mg/kg of Formula Ia with 5 mg/kg of docetaxelsynergized to inhibit LuCap 35 V human primary prostate tumor growth invivo greater than Formula Ia or docetaxel alone and resulted in tumorregression.

FIG. 49 shows the mean tumor volume change over time with female SCIDBeige nude mice with LuCap 35V human primary prostate cancer tumor cellxenografts dosed on day 0 with: docetaxel 5 mg/kg intravenously on days1, 5 and 9 (q4d×3) Formula Ib 2.5 mg/kg orally daily for 15 days,Formula Ib 5 mg/kg orally daily for 15 days and the combinations of:docetaxel 5 mg/kg intravenously on days 1, 5 and 9 (q4d×3) and FormulaIb 2.5 mg/kg orally daily for 15 days and docetaxel 5 mg/kgintravenously on days 1, 5 and 9 (q4d×3) and Formula Ib 5 mg/kg orallydaily for 15 days along with mice receiving no drug (Vehicle group). Thecombination of 2.5 mg/kg of Formula Ib with 5 mg/kg of docetaxelsynergized to inhibit LuCap 35 V human primary prostate tumor growth invivo greater than Formula Ib or docetaxel alone and resulted in tumorregression. The combination of 5.0 mg/kg of Formula Ib with 5 mg/kg ofdocetaxel also synergized to inhibit LuCap 35 V human primary prostatetumor growth in vivo greater than Formula Ib or docetaxel alone andresulted in tumor regression.

FIG. 50 shows the mean tumor volume change over time with CRL femalenu/nu mice with PC3-NCI human primary prostate cancer tumor cellxenografts dosed on days 1, 5, 9, and 13 (q4d×4) with 2.5 mg/kgdocetaxel intravenously, Formula 1b 2.5 mg/kg Formula 1b on days 1, 5, 9and 13 (q4d×4), Formula Ib 10 mg/kg orally on days 1, 5, 9 and 13(q4d×4), and the combination of: docetaxel 2.5 mg/kg intravenoulsy andFormula Ib 10 mg/kg orally, along with mice receiving no drug (Vehiclegroup). The combination of 10 mg/kg of Formula Ib dosed on days 1, 5, 9and 13 with 5 mg/kg of docetaxel synergized to inhibit PC3-NCI humanprimary prostate tumor growth in vivo greater than Formula Ib ordocetaxel alone and resulted in tumor regression.

FIG. 51 shows the mean tumor volume change over time with CRL femalenu/nu mice with PC3-NCI human primary prostate cancer cell xenograftsdosed on day 0 with: gemcitabine 100 mg/kg intraperitonealy every 3 days(q3d) for 4 times, Formula Ia (GDC-0941) 150 mg/kg orally every 3 days(q3d) for 4 times Formula Ib 2.5 mg/kg orally every 3 (q3d) days for 4times, Formula Ib 5 mg/kg orally every 3 days for 4 times and thecombinations of: gemcitabine 100 mg/kg intraperitonealy every 3 days for4 times and Formula Ia 150 mg/kg orally every 3 days for 4 times;gemcitabine 100 mg/kg intraperitonealy every 3 days for 4 times andFormula Ib 2.5 mg/kg orally every 3 days for 4 times, gemcitabine 100mg/kg intraperitonealy every 3 days for 4 times and Formula Ib 5 mg/kgorally every 3 days for 4 times, gemcitabine 100 mg/kg intraperitonealyevery 3 days for 4 times and Formula Ib 10 mg/kg orally every 3 days for4 times, along with mice receiving no drug (Vehicle group). Thecombination of 150 mg/kg of Formula Ia dosed orally every 3 days (q3d)for 4 times with 100 mg/kg of gemcitabine synergized to inhibit PC3-NCIhuman primary prostate tumor growth in vivo greater than Formula Ia orgemcitabine alone and resulted in tumor regression and tumor growthdelay. The combination of 2.5 mg/kg of Formula Ib dosed orally every 3days (q3d) for 4 times with 100 mg/kg of gemcitabine synergized toinhibit PC3-NCI human primary prostate tumor growth in vivo greater thanFormula Ia or gemcitabine alone and resulted in tumor regression andtumor growth delay. The combination of 5.0 mg/kg of Formula Ib dosedorally every 3 days (q3d) for 4 times with 100 mg/kg of gemcitabinesynergized to inhibit PC3-NCI human primary prostate tumor growth invivo greater than Formula Ia or gemcitabine alone and resulted in tumorregression and tumor growth delay. The combination of 10 mg/kg ofFormula Ib dosed orally every 3 days (q3d) for 4 times with 100 mg/kg ofgemcitabine synergized to inhibit PC3-NCI human primary prostate tumorgrowth in vivo greater than Formula Ia or gemcitabine alone and resultedin tumor regression and tumor growth delay.

FIG. 52 shows the mean tumor volume change over time with Harlan femalenude mice with NCI-H2122 (K-ras) non-small cell lung cancer (NSCLC)tumor cell xenografts dosed on day 0 with: PD-0325901 6.3 mg/kg orallydaily for 21 days, Formula Ia (GDC-0941) at 100 mg/kg orally daily for21 days, and the combination of: PD-0325901 6.3 mg/kg orally daily for21 days and Formula Ia at 100 mg/kg orally daily for 21 days, along withmice receiving no drug (Vehicle group). The combination of 100 mg/kg ofFormula Ia with 6.3 mg/kg of PD-0325901 synergized to inhibit NCI-H2122(K-ras) NSCLC tumor growth in vivo greater than Formula Ia or PD-0325901alone and resulted in tumor regression.

Genetically Engineered Mouse Model Tumor Efficacy

Combination therapy with Formula I compounds and chemotherapeutic agentswas effective in treatment of small cell lung cancer (SCLC), non-smallcell lung cancer (NSCLC), and pancreatic adenocarcinoma (PDAC) ingenetically engineered mouse models (GEMM) which recapitulate humantumor progression in an endogenous tissue micro-environment (Singh, M.and Johnson, L. (2006) Clin. Cancer Res. 12(18):5312-5328; US2007/0292948, both of which are incorporated by reference in theirentirety). The surprising and unexpected results from these experimentsmay predict the clinical response of certain patient populations havingselect tumor types and mutations to such combination therapy withFormula I and II compounds and chemotherapeutic agents.

In a model for SCLC (Meuwissen et al (2003) Cancer Cell 4(3):181-189)bearing the mutations commonly found in the patient population, FormulaIa compound alone did not show effects on tumor growth as assessed bymicroCT imaging but did have an effect (significant hazard ratio) onsurvival as compared to controls. Animals dosed with the combination ofFormula Ia compound and mB20-4.1.1 murine anti-VEGF-A showed asignificant inhibition and regression of tumor growth by microCTimaging, when compared to controls and each single drug. The combinationof Formula Ia compound and mB20-4.1 murine anti-VEGF-A also had astatistically significant impact on overall survival compared tocontrols. Animals dosed with the triple combination of Formula Iacompound, carboplatin, and mB20-4.1.1 murine anti-VEGF-A also showed ameasurable and durable anti-tumor response as compared to single agents.The triple combination significantly increased survival as compared tosingle agents, and Formula Ia compound in this triple regimen enhancedthe survival advantage compared to the carboplatin and mB20-4.1 murineanti-VEGF-A dual combination. In addition, these combinatorial regimensmarkedly decreased the incidence of metastases to regional lymph nodesand the liver when compared to controls and each single drug.

A GEMM for pancreatic cancer (Aguirre et al. (2003) Genes & Development17:3112-3126) encompasses the mutations found in a majority of patientswith this disease. These mice, when treated with Formula Ia compound,showed an initial decrease in tumor growth via ultrasound as compared tocontrol-treated mice. However, this response was not durable and thesingle agent treatment had a modest (not statistically significant)effect on survival in these mice. In contrast, Formula Ia in combinationwith mB20-4.1.1 murine anti-VEGF-A showed significant decreases in tumorgrowth via ultrasound (as compared to untreated mice and single agents)as well as a significant impact on survival. Combination therapy withgemcitabine, either with or without mB20-4.1.1 murine anti-VEGF-A, didnot show significant improvements in tumor growth inhibition (viaultrasound) or survival as compared to gemcitabine alone.

In a K-ras-driven NSCLC GEMM (Johnson et al (2001) Nature 410:1111-1116;Jackson et al (2001) Genes & Development 15:3243-3248), Formula Iacompound treatment alone minimally affected tumor growth as measured bymicroCT imaging, and had no effect on survival as compared to controls.Treatment with Formula Ia compound in combination with erlotinib showedmodest tumor growth inhibition and survival advantage as compared to thesingle agent treatments. Treatment with Formula Ia compound incombination with mB20-4.1.1 murine anti-VEGF-A resulted in a notabledecrease in tumor growth and increase in survival as compared tocontrols, albeit not significantly greater than the effects observedwith mB20-4.1.1 murine anti-VEGF-A as a single agent. Animals dosed withthe triple combination of Formula Ia compound, carboplatin, andmB20-4.1.1 murine anti-VEGF-A also showed a measurable and durableanti-tumor response as compared to controls and also markedly impactedsurvival in this model.

Pharmaceutical Compositions

Pharmaceutical compositions or formulations of the present inventioninclude combinations of Formula I or II compounds, a chemotherapeuticagent, and one or more pharmaceutically acceptable carrier, glidant,diluent, or excipient.

The Formula I or II compounds, and chemotherapeutic agents of thepresent invention may exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike, and it is intended that the invention embrace both solvated andunsolvated forms.

The Formula I or II compounds, and chemotherapeutic agents of thepresent invention may also exist in different tautomeric forms, and allsuch forms are embraced within the scope of the invention. The term“tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Pharmaceutical compositions encompass both the bulk composition andindividual dosage units comprised of more than one (e.g., two)pharmaceutically active agents including a Formula I or II compound anda chemotherapeutic agent selected from the lists of the additionalagents described herein, along with any pharmaceutically inactiveexcipients, diluents, carriers, or glidants. The bulk composition andeach individual dosage unit can contain fixed amounts of the aforesaidpharmaceutically active agents. The bulk composition is material thathas not yet been formed into individual dosage units. An illustrativedosage unit is an oral dosage unit such as tablets, pills, capsules, andthe like. Similarly, the herein-described method of treating a patientby administering a pharmaceutical composition of the present inventionis also intended to encompass the administration of the bulk compositionand individual dosage units.

Pharmaceutical compositions also embrace isotopically-labeled compoundsof the present invention which are identical to those recited herein,but for the fact that one or more atoms are replaced by an atom havingan atomic mass or mass number different from the atomic mass or massnumber usually found in nature. All isotopes of any particular atom orelement as specified are contemplated within the scope of the compoundsof the invention, and their uses. Exemplary isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine,chlorine and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O,¹⁸O, ³²P, ³³P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I and ¹²⁵I. Certainisotopically-labeled compounds of the present invention (e.g., thoselabeled with ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (³H) and carbon-14 (¹⁴C) isotopes areuseful for their ease of preparation and detectability. Further,substitution with heavier isotopes such as deuterium (²H) may affordcertain therapeutic advantages resulting from greater metabolicstability (e.g., increased in vivo half-life or reduced dosagerequirements) and hence may be preferred in some circumstances. Positronemitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸F are useful for positronemission tomography (PET) studies to examine substrate receptoroccupancy. Isotopically labeled compounds of the present invention cangenerally be prepared by following procedures analogous to thosedisclosed in the Schemes and/or in the Examples herein below, bysubstituting an isotopically labeled reagent for a non-isotopicallylabeled reagent.

Formula I or II compounds and chemotherapeutic agents are formulated inaccordance with standard pharmaceutical practice for use in atherapeutic combination for therapeutic treatment (includingprophylactic treatment) of hyperproliferative disorders in mammalsincluding humans. The invention provides a pharmaceutical compositioncomprising a Formula I or II compound in association with one or morepharmaceutically acceptable carrier, glidant, diluent, or excipient.

Suitable carriers, diluents and excipients are well known to thoseskilled in the art and include materials such as carbohydrates, waxes,water soluble and/or swellable polymers, hydrophilic or hydrophobicmaterials, gelatin, oils, solvents, water and the like. The particularcarrier, diluent or excipient used will depend upon the means andpurpose for which the compound of the present invention is beingapplied. Solvents are generally selected based on solvents recognized bypersons skilled in the art as safe (GRAS) to be administered to amammal. In general, safe solvents are non-toxic aqueous solvents such aswater and other non-toxic solvents that are soluble or miscible inwater. Suitable aqueous solvents include water, ethanol, propyleneglycol, polyethylene glycols (e.g., PEG 400, PEG 300), etc. and mixturesthereof. The formulations may also include one or more buffers,stabilizing agents, surfactants, wetting agents, lubricating agents,emulsifiers, suspending agents, preservatives, antioxidants, opaquingagents, glidants, processing aids, colorants, sweeteners, perfumingagents, flavoring agents and other known additives to provide an elegantpresentation of the drug (i.e., a compound of the present invention orpharmaceutical composition thereof) or aid in the manufacturing of thepharmaceutical product (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a Formula I or II compound having the desired degree of puritymay optionally be mixed with pharmaceutically acceptable diluents,carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences(1995) 18th edition, Mack Publ. Co., Easton, Pa.), in the form of alyophilized formulation, milled powder, or an aqueous solution.Formulation may be conducted by mixing at ambient temperature at theappropriate pH, and at the desired degree of purity, withphysiologically acceptable carriers, i.e., carriers that are non-toxicto recipients at the dosages and concentrations employed. The pH of theformulation depends mainly on the particular use and the concentrationof compound, but may range from about 3 to about 8.

The pharmaceutical formulation is preferably sterile. In particular,formulations to be used for in vivo administration must be sterile. Suchsterilization is readily accomplished by filtration through sterilefiltration membranes.

The pharmaceutical formulation ordinarily can be stored as a solidcomposition, a lyophilized formulation or as an aqueous solution.

The pharmaceutical formulations of the invention will be dosed andadministered in a fashion, i.e., amounts, concentrations, schedules,course, vehicles and route of administration, consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat the coagulation factor mediated disorder. Suchamount is preferably below the amount that is toxic to the host orrenders the host significantly more susceptible to bleeding.

As a general proposition, the initial pharmaceutically effective amountof the Formula I or II compound administered orally or parenterally perdose will be in the range of about 0.01-1000 mg/kg, namely about 0.1 to20 mg/kg of patient body weight per day, with the typical initial rangeof compound used being 0.3 to 15 mg/kg/day. The dose of the Formula I orII compound and the dose of the chemotherapeutic agent to beadministered may range for each from about 1 mg to about 1000 mg perunit dosage form, or from about 10 mg to about 100 mg per unit dosageform. The doses of Formula I or II compound and the chemotherapeuticagent may administered in a ratio of about 1:50 to about 50:1 by weight,or in a ratio of about 1:10 to about 10:1 by weight.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate)microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 18th edition, (1995) Mack Publ. Co.,Easton, Pa.

Sustained-release preparations of Formula I and II compounds may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D(−)3-hydroxybutyric acid.

The pharmaceutical formulations include those suitable for theadministration routes detailed herein. The formulations may convenientlybe presented in unit dosage form and may be prepared by any of themethods well known in the art of pharmacy. Techniques and formulationsgenerally are found in Remington's Pharmaceutical Sciences 18^(th) Ed.(1995) Mack Publishing Co., Easton, Pa. Such methods include the step ofbringing into association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations of a compound of Formula I or II and/or chemotherapeuticagent suitable for oral administration may be prepared as discrete unitssuch as pills, hard or soft e.g., gelatin capsules, cachets, troches,lozenges, aqueous or oil suspensions, dispersible powders or granules,emulsions, syrups or elixirs each containing a predetermined amount of acompound of Formula I or II and/or a chemotherapeutic agent. The amountof compound of Formula I or II and the amount of chemotherapeutic agentmay be formulated in a pill, capsule, solution or suspension as acombined formulation. Alternatively, the Formula I or II compound andthe chemotherapeutic agent may be formulated separately in a pill,capsule, solution or suspension for administration by alternation.

Formulations may be prepared according to any method known to the artfor the manufacture of pharmaceutical compositions and such compositionsmay contain one or more agents including sweetening agents, flavoringagents, coloring agents and preserving agents, in order to provide apalatable preparation. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom.

Tablet excipients of a pharmaceutical formulation of the invention mayinclude: Filler (or diluent) to increase the bulk volume of the powdereddrug making up the tablet; Disintegrants to encourage the tablet tobreak down into small fragments, ideally individual drug particles, whenit is ingested and promote the rapid dissolution and absorption of drug;Binder to ensure that granules and tablets can be formed with therequired mechanical strength and hold a tablet together after it hasbeen compressed, preventing it from breaking down into its componentpowders during packaging, shipping and routine handling; Glidant toimprove the flowability of the powder making up the tablet duringproduction; Lubricant to ensure that the tableting powder does notadhere to the equipment used to press the tablet during manufacture.They improve the flow of the powder mixes through the presses andminimize friction and breakage as the finished tablets are ejected fromthe equipment; Antiadherent with function similar to that of theglidant, reducing adhesion between the powder making up the tablet andthe machine that is used to punch out the shape of the tablet duringmanufacture; Flavor incorporated into tablets to give them a morepleasant taste or to mask an unpleasant one, and Colorant to aididentification and patient compliance.

Tablets containing the active ingredient in admixture with non-toxicpharmaceutically acceptable excipient which are suitable for manufactureof tablets are acceptable. These excipients may be, for example, inertdiluents, such as calcium or sodium carbonate, lactose, calcium orsodium phosphate; granulating and disintegrating agents, such as maizestarch, or alginic acid; binding agents, such as starch, gelatin oracacia; and lubricating agents, such as magnesium stearate, stearic acidor talc. Tablets may be uncoated or may be coated by known techniquesincluding microencapsulation to delay disintegration and adsorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base.

If desired, the aqueous phase of the cream base may include a polyhydricalcohol, i.e., an alcohol having two or more hydroxyl groups such aspropylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol andpolyethylene glycol (including PEG 400) and mixtures thereof. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active ingredient through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethyl sulfoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner, including a mixture of atleast one emulsifier with a fat or an oil, or with both a fat and anoil. Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. Together, theemulsifier(s) with or without stabilizer(s) make up an emulsifying wax,and the wax together with the oil and fat comprise an emulsifyingointment base which forms the oily dispersed phase of creamformulations. Emulsifiers and emulsion stabilizers suitable for use inthe formulation of the invention include Tween® 60, Span® 80,cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glycerylmono-stearate and sodium lauryl sulfate.

Aqueous suspensions of the pharmaceutical formulations of the inventioncontain the active materials in admixture with excipients suitable forthe manufacture of aqueous suspensions. Such excipients include asuspending agent, such as sodium carboxymethylcellulose, croscarmellose,povidone, methylcellulose, hydroxypropyl methylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, anddispersing or wetting agents such as a naturally occurring phosphatide(e.g., lecithin), a condensation product of an alkylene oxide with afatty acid (e.g., polyoxyethylene stearate), a condensation product ofethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Pharmaceutical compositions may be in the form of a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension may be formulated according to the known artusing those suitable dispersing or wetting agents and suspending agentswhich have been mentioned above. The sterile injectable preparation maybe a solution or a suspension in a non-toxic parenterally acceptablediluent or solvent, such as a solution in 1,3-butanediol or preparedfrom a lyophilized powder. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile fixed oils may conventionally beemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid may likewise be used in thepreparation of injectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

Formula I and II compounds may be employed in combination with otherchemotherapeutic agents for the treatment of a hyperproliferativedisease or disorder, including tumors, cancers, and neoplastic tissue,along with pre-malignant and non-neoplastic or non-malignanthyperproliferative disorders. In certain embodiments, a compound ofFormula I or II is combined in a pharmaceutical combination formulation,or dosing regimen as combination therapy, with a second compound thathas anti-hyperproliferative properties or that is useful for treatingthe hyperproliferative disorder. The second compound of thepharmaceutical combination formulation or dosing regimen preferably hascomplementary activities to the compound of Formula I or II, and suchthat they do not adversely affect each other. Such compounds may beadministered in amounts that are effective for the purpose intended. Inone embodiment, a pharmaceutical formulation of this invention comprisesa compound of Formula I or II, or a stereoisomer, geometric isomer,tautomer, solvate, metabolite, or pharmaceutically acceptable saltthereof, in combination with a chemotherapeutic agent such as describedherein. In another embodiment, the therapeutic combination isadministered by a dosing regimen wherein the therapeutically effectiveamount of a compound having Formula I or II is administered in a rangefrom twice daily to once every three weeks (q3wk), and thetherapeutically effective amount of the chemotherapeutic agent isadministered in a range from twice daily to once every three weeks.

Therapeutic combinations of the invention include a product comprising acompound having Formula I or II, and a chemotherapeutic agent selectedfrom erlotinib, docetaxel, 5-FU, gemcitabine, PD-0325901, cisplatin,carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab,temozolomide, tamoxifen, doxorubicin, Akti-1/2, HPPD, rapamycin, andlapatinib as a combined preparation for separate, simultaneous orsequential use in the treatment of a hyperproliferative disorder.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments, such as to increase the therapeutic index or mitigatetoxicity or other side-effects or consequences.

In a particular embodiment of anti-cancer therapy, a compound of FormulaI or II, or a stereoisomer, geometric isomer, tautomer, solvate,metabolite, or pharmaceutically acceptable salt thereof, may be combinedwith a chemotherapeutic agent, including hormonal or antibody agentssuch as those described herein, as well as combined with surgicaltherapy and radiotherapy. Combination therapies according to the presentinvention thus comprise the administration of at least one compound ofFormula I or II, or a stereoisomer, geometric isomer, tautomer, solvate,metabolite, or pharmaceutically acceptable salt thereof, and the use ofat least one other cancer treatment method. The amounts of thecompound(s) of Formula I or II and the other pharmaceutically activechemotherapeutic agent(s) and the relative timings of administrationwill be selected in order to achieve the desired combined therapeuticeffect.

Administration of Pharmaceutical Compositions

The compounds of the invention may be administered by any routeappropriate to the condition to be treated. Suitable routes includeoral, parenteral (including subcutaneous, intramuscular, intravenous,intraarterial, inhalation, intradermal, intrathecal, epidural, andinfusion techniques), transdermal, rectal, nasal, topical (includingbuccal and sublingual), vaginal, intraperitoneal, intrapulmonary andintranasal. Topical administration can also involve the use oftransdermal administration such as transdermal patches or iontophoresisdevices. Formulation of drugs is discussed in Remington's PharmaceuticalSciences, 18^(th) Ed., (1995) Mack Publishing Co., Easton, Pa. Otherexamples of drug formulations can be found in Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol 3,2^(nd) Ed., New York, N.Y. For local immunosuppressive treatment, thecompounds may be administered by intralesional administration, includingperfusing or otherwise contacting the graft with the inhibitor beforetransplantation. It will be appreciated that the preferred route mayvary with for example the condition of the recipient. Where the compoundis administered orally, it may be formulated as a pill, capsule, tablet,etc. with a pharmaceutically acceptable carrier, glidant, or excipient.Where the compound is administered parenterally, it may be formulatedwith a pharmaceutically acceptable parenteral vehicle or diluent, and ina unit dosage injectable form, as detailed below.

A dose to treat human patients may range from about 10 mg to about 1000mg of Formula I or II compound. A typical dose may be about 100 mg toabout 300 mg of the compound. A dose may be administered once a day(QD), twice per day (BID), or more frequently, depending on thepharmacokinetic (PK) and pharmacodynamic (PD) properties, includingabsorption, distribution, metabolism, and excretion of the particularcompound. In addition, toxicity factors may influence the dosage andadministration dosing regimen. When administered orally, the pill,capsule, or tablet may be ingested twice daily, daily or less frequentlysuch as weekly or once every two or three weeks for a specified periodof time. The regimen may be repeated for a number of cycles of therapy.

Methods of Treatment

Therapeutic combinations of: (1) a Formula I or II compound and (2) achemotherapeutic agent are useful for treating diseases, conditionsand/or disorders including, but not limited to, those characterized byactivation of the PI3 kinase pathway. Accordingly, another aspect ofthis invention includes methods of treating diseases or conditions thatcan be treated by inhibiting lipid kinases, including PI3. In oneembodiment, the method comprises administering to a mammal in needthereof a therapeutically effective amount of a compound of Formula I orII, or a stereoisomer, geometric isomer, tautomer, solvate, metabolite,or pharmaceutically acceptable salt thereof. Therapeutic combinations of(1) a Formula I or II compound and (2) a chemotherapeutic agent may beemployed for the treatment of a hyperproliferative disease or disorder,including tumors, cancers, an dneoplastic tissue, along withpre-malignant and non-neoplastic or non-malignant hyperproliferativedisorders. In one embodiment, a human patient is treated with atherapeutic combination and a pharmaceutically acceptable carrier,adjuvant, or vehicle, wherein the Formula I or II compound, ormetabolite thereof, of said therapeutic combination is present in anamount to detectably inhibit PI3 kinase activity.

Cancers which can be treated according to the methods of this inventioninclude, but are not limited to, breast, ovary, cervix, prostate,testis, genitourinary tract, esophagus, larynx, glioblastoma,neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoidcarcinoma, large cell carcinoma, non-small cell lung carcinoma (NSCLC),small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma,pancreas, adenocarcinoma, thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidneycarcinoma, myeloid disorders, lymphoid disorders, hairy cells, buccalcavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine,colon-rectum, large intestine, rectum, brain and central nervous system,Hodgkin's and leukemia.

Another aspect of this invention provides a pharmaceutical compositionor therapeutic combination for use in the treatment of the diseases orconditions described herein in a mammal, for example, a human, sufferingfrom such disease or condition. Also provided is the use of apharmaceutical composition in the preparation of a medicament for thetreatment of the diseases and conditions described herein in awarm-blooded animal, such as a mammal, for example a human, sufferingfrom such disorder.

Metabolites of Compounds of Formula I and II

Also falling within the scope of this invention are the in vivometabolic products of Formula I and II described herein. Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, deamidation, esterification, deesterification, enzymaticcleavage, and the like, of the administered compound. Accordingly, theinvention includes metabolites of compounds of Formula I and II,including compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing Formula I and II compounds useful for the treatment ofthe diseases and disorders described above is provided. In oneembodiment, the kit comprises a container comprising a compound ofFormula I, or a stereoisomer, geometric isomer, tautomer, solvate,metabolite, or pharmaceutically acceptable salt thereof. The kit mayfurther comprise a label or package insert, on or associated with thecontainer. The term “package insert” is used to refer to instructionscustomarily included in commercial packages of therapeutic products,that contain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch therapeutic products. Suitable containers include, for example,bottles, vials, syringes, blister pack, etc. The container may be formedfrom a variety of materials such as glass or plastic. The container mayhold a compound of Formula I or II or a formulation thereof which iseffective for treating the condition and may have a sterile access port(for example, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is a compound of Formula I or II.The label or package insert indicates that the composition is used fortreating the condition of choice, such as cancer. In one embodiment, thelabel or package inserts indicates that the composition comprising acompound of Formula I or II can be used to treat a disorder resultingfrom abnormal cell growth. The label or package insert may also indicatethat the composition can be used to treat other disorders.Alternatively, or additionally, the article of manufacture may furthercomprise a second container comprising a pharmaceutically acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

The kit may further comprise directions for the administration of thecompound of Formula I or II and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I or II and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I or II, such as tablets orcapsules. Such a kit preferably includes a number of unit dosages. Suchkits can include a card having the dosages oriented in the order oftheir intended use. An example of such a kit is a “blister pack”.Blister packs are well known in the packaging industry and are widelyused for packaging pharmaceutical unit dosage forms. If desired, amemory aid can be provided, for example in the form of numbers, letters,or other markings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I or II contained therein; and optionally (b)a second container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Where the kit comprises a composition of Formula I or II and a secondtherapeutic agent, i.e. the chemotherapeutic agent, the kit may comprisea container for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

GENERAL PREPARATIVE PROCEDURES General Procedure A-1 Suzuki Coupling

The Suzuki-type coupling reaction is useful to attach a fused bicyclicheterocycle or heteroaryl at the 2-position of the pyrimidine ring (seeScheme 4). Generally, substituted2-chloro-4-morpholinothieno[3,2-d]pyrimidine 5 or substituted2-chloro-4-morpholinothieno[2,3-d]pyrimidine 6 may be combined with 1.5equivalents of4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1H-indazole 7, anddissolved in 3 equivalents of sodium carbonate as a 1 molar solution inwater and an equal volume of acetonitrile. A catalytic amount, or more,of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. A variety ofboronic acids or boronic esters can be used in place of the indazoleboronic ester indicated. Also alternatively, the nitrogen of theindazole may be protected, for example with a tetrahydropyranyl group.See compound 40. In some cases potassium acetate was used in place ofsodium carbonate to adjust the pH of the aqueous layer. The reaction wasthen heated to about 140-150° C. under pressure in a Biotage Optimizermicrowave reactor (Biotage, Inc.) for 10 to 30 minutes. The contents areextracted with ethyl acetate, or another organic solvent. Afterevaporation of the organic layer the product, 8 or 9, may be purified onsilica or by reverse phase HPLC.

General Procedure A-2 Suzuki Coupling

The Suzuki-type coupling reaction is useful to attach a monocyclicheteroaryl at the 2-position of the pyrimidine ring (see Scheme 4).Generally, substituted 2-chloro-4-morpholinothieno[3,2-d]pyrimidine 5 orsubstituted 2-chloro-4-morpholinothieno[2,3-d]pyrimidine 6 may becombined with 1.5 equivalents of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine 7a, anddissolved in 3 equivalents of sodium or potassium carbonate as a 1 molarsolution in water and an equal volume of acetonitrile. A catalyticamount, or more, of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. A variety ofboronic acids or boronic esters can be used in place of the pinacolboronic ester indicated. Also alternatively, the nitrogen of thepyrimidin-2-amine may be protected, for example with a tetrahydropyranylgroup. In some cases potassium acetate was used in place of sodiumcarbonate to adjust the pH of the aqueous layer. The reaction was thenheated, for example to about 100-150° C. under pressure in a BiotageOptimizer microwave reactor (Biotage, Inc.) for 10 to 30 minutes. Thecontents are extracted with ethyl acetate, or another organic solvent.After evaporation of the organic layer the product, 8a or 9a, may bepurified on silica or by reverse phase HPLC.

General Procedure B Amide Coupling

2-(1H-Indazol-4-yl)-4-morpholinothieno[3,2-d]pyrimidine-6-carboxylicacid 13 or2-(1H-indazol-4-yl)-4-morpholinothieno[2,3-d]pyrimidine-6-carboxylicacid 14 is treated with 1.5 eq HATU, 3 eq of alkylamine and 3 eq ofDIPEA in DMF to approximately 0.1 M concentration. The reaction isstirred until complete and extracted in ethylacetate with saturatedbicarbonate solution one time. The organic layer is dried, filtered andconcentrated to yield the crude intermediate. This intermediate ispurified via reverse phase HPLC to yield product 15 or 16.

General Procedure B-1 Amide Coupling

4-Morpholino-2-(pyridin-3-yl)thieno[3,2-d]pyrimidine-6-carboxylic acid13a or 4-morpholino-2-(pyridin-3-yl)thieno[2,3-d]pyrimidine-6-carboxylicacid 14a is treated with 1.5 eq HATU, 3 eq of an alkylamine (R—NH₂) and3 eq of DIPEA in DMF to approximately 0.1 M concentration. The reactionis stirred until complete and extracted in ethylacetate with saturatedbicarbonate solution one time. The organic layer is dried, filtered andconcentrated to yield the crude intermediate. This intermediate ispurified via reverse phase HPLC to yield product 15a or 16a.

General Procedure B-2 Amide Coupling

2-Chloro-4-morpholino-6-((piperazin-1-yl)methyl)thieno[3,2-d]pyrimidineor2-chloro-4-morpholino-6-((piperazin-1-yl)methyl)thieno[2,3-d]pyrimidineis treated with 1.5 eq HATU, 3 eq of carboxylic acid (RCO₂H) and 3 eq ofDIPEA in DMF to approximately 0.1 M concentration. The reaction isstirred until complete and extracted in ethyl acetate with saturatedbicarbonate solution one time. The organic layer is dried, filtered andconcentrated to yield the crude intermediate.

General Procedure B-3 Reductive Amination

2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde 10 or2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde 33 wasdissolved to a 0.2 M concentration in dichloroethane. To this solutionwas added 1.5 to 2.0 equivalents of an amine (R¹R²NH), 10 equivalents oftrimethylorthoformate, and 1 equivalent of acetic acid. The mixture wasallowed to stir for 2-6 hours prior to adding 1.5 equivalents of sodiumtriacetoxyborohydride. Following 12 to 16 hours of stirring the reactionwas poured into saturated sodium bicarbonate and extracted several timeswith ethyl acetate to give the reductive amination intermediate whichwas either purified on silica gel or used crude in the next reaction.

General Procedure C Sulfonamide Formation

2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-sulfonyl chloride 17 wassuspended in 1 mL of DCM before addition of 2 eq of amine and 3 eq ofDIPEA. The reactions were monitored by LCMS until complete. The crudereaction mixtures were diluted with ethyl acetate, extracted withsaturated ammonium chloride and back-extracted once with ethyl acetate.The organic layers were combined and concentrated to dryness. The crudesulfonamide intermediates 18 were used directly in the subsequent Suzukicouplings.

General Procedure D Alcohol Synthesis

2-Chloro-4-morpholinothieno[3,2-d]pyrimidine 4 was suspended to a 0.2molar concentration in THF and cooled to −50° C. in a dryice/acetonitrile bath before adding 2 equivalents of 2.5 M nBuLi inhexanes. After 15 min 3.0 molar equivalents of a cyclic or acyclicketone was added to the solution. The reaction continued to stir at −50°C. for 1 h and then in most cases was allowed to come to 0° C. When thereaction was complete by TLC or mass spec. it was quenched into asaturated ammonium chloride solution and extracted two times with EtOAc.The organic layer was concentrated and either used as a crude mixture,purified on silica, or the product 12 could be dissolved in a minimalamount of acetonitrile and filtered to remove remaining startingmaterial 4.

General Procedure E Removal of t-butoxylcarbonyl (BOC) Group

Ten or more equivalents of 4N HCl in dioxane, with or withoutdichloromethane as a co-solvent, are added to the starting material(general scheme shown above but similar scaffolds also used). Heating upto 40° C. for several hours is occasionally required to remove the Bocgroup. The reaction is concentrated to dryness and may be used crude insubsequent reactions.

General Procedure F Suzuki Coupling Reactions in One Pot

2-Chloro-6-iodo-4-morpholinothieno[3,2-d]pyrimidine 19 (1 eq),phenylboronic acid or heterocycleboronic acid (R¹—B(OH)₂, 1.1 eq) andbis(triphenylphosphine)palladium(II) dichloride (0.1 eq) in 1M Na₂CO₃aqueous solution (3 eq) and acetonitrile (3 eq) was heated to 100° C. ina sealed microwave reactor for 10 to 40 min to give 5. Upon completion,4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole 7 (1.3 eq)and bis(triphenylphosphine)palladium(II) dichloride (0.1 eq) were addedin the same pot. The reaction mixture was heated to 150° C. in a sealedmicrowave reactor for 10 to 15 min. The mixture was extracted with ethylacetate (3×5 mL). The combined organic layers were concentrated to yieldcrude 8.

General Procedure G Amide Coupling Reaction

2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-6-amine 22 (1 eq), Acidchloride (1.5˜2 eq) and triethylamine (2 eq) in dichloromethane wasstirred. The reaction was monitored by LC/MS until complete. The mixturewas evaporated to give the crude amide 23, which was directly used forthe next step reaction without purification.

General Procedure H Preparation of Acetamide, Benzamidines, andSulfonamides

To a 0.25 to 0.40 M solution of1-(2-chloro-4-morpholinothieno[2,3-d]pyrimidin-6-yl)-N-methylmethanaminein DCM cooled to 0° C. was added 1.5 eq. of TEA, followed by thedrop-wise addition of 1.0 to 1.5 eq. of an alkyl or aryl-acid chlorideor a sulfonylchloride, diluted in DCM. The reaction is stirred atambient temperature and monitored for completeness by LCMS. Aftercompletion, the reaction volume is increased with DCM, and diluteaqueous sodium bicarbonate is added to the solution. The organic andaqueous layers are separated. Finally, the organic layer is washed withbrine and dried (MgSO₄). The dried organic solution is concentrated invacuo and the product is purified by silica chromatography if necessary.

General Procedure I Amide Coupling Reaction for Benzenamine

3-(2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)benzenamine 24 (1eq), carboxylic acid (1.5 eq), 1-hydroxy-7-azabenzotriazole (0.2 eq),O-(7-azabenzotriazol-1-yl)-(N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 1.5 eq), and N,N-diisopropylethylamine (2.5eq) in DMF was stirred at room temperature. The reaction was monitoredby LC/MS until complete. The reaction mixture was diluted with ethylacetate, washed with saturated sodium bicarbonate and brine. The organiclayer was dried over MgSO₄, filtered and evaporated to yield amideproduct 25.

General Procedure J 6-Iodo Displacement and 2-Suzuki Coupling

To a solution of 2-chloro-6-iodo-4-morpholinothieno[3,2-d]pyrimidine 19(0.05 g, 0.13 mmol) in DMF (1.00 mL) was added the appropriate aniline(200 mol %), Cs—₂CO₃ (50 mol %), Pd₂(dba)₃ (5 mol %), and XANTPHOS (10mol %). The reaction was heated to 110° C. under pressure in a Biotageoptimizer microwave reactor for 30 min. The resulting solution wasconcentrated in vacuo to give 26, after following General Procedure A.

General Procedure K 6-Aminoalkyl Acylation and 2-Suzuki Coupling

To a solution of(2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanamine 27 (50 mg,0.2 mmol) in CH₂Cl₂ (4 mL) was added Et₃N (84 μL, 0.6 mmol) and theappropriate acid chloride or HCl salt thereof (0.3 mmol). The reactionstirred 18-48 hr at room temperature before being quenched with water.The aqueous layer was extracted with EtOAc. The combined organics weredried over Na₂SO₄ and concentrated in vacuo. The 2-chloro crude productwas coupled with boronate reagent 7 and palladium catalyst according toGeneral Procedure A to give 28 which was purified by reversed phase HPLCpurification.

Alternatively, to a solution of(2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanamine 27 (111mg, 0.39 mmol) in DMF (5 mL) was added 2,6-lutidine (48.2 μL, 0.41 mmol)and the appropriate acid chloride or HCl salt thereof (0.39 mmol). Thereaction stirred 18-72 hr at room temperature before being quenched withwater. The aqueous layer was extracted with EtOAc. The combined organicswere dried over MgSO₄ and concentrated in vacuo. The 2-chloro crudeproduct was coupled with boronate reagent 7 and palladium catalystaccording to General Procedure A to give 20 mg of 28 which was purifiedby reversed phase HPLC purification.

General Procedure L Amine Substitution on Fluoropyridine

A mixture of2-chloro-6-(6-fluoropyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidine or2-chloro-6-(6-fluoropyridin-3-yl)-4-morpholinothieno[2,3-d]pyrimidinecompound, about four equivalents of a primary or secondary amine (R═H,C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀heterocyclyl, C₆-C₂₀ aryl, or C₁-C₂₀ heteroaryl), and about two eq.diisopropylethylamine in N-methylpyrrolidine (˜0.1M) is heated to about130-140° C. in a sealed microwave reactor for 10˜40 min, followed byremoval of volatiles under high vacuum. The crude mixture is purified byflash chromatography to give intermediate2-chloro-6-(6-aminopyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidine or2-chloro-6-(6-aminopyridin-3-yl)-4-morpholinothieno[2,3-d]pyrimidinecompound, which may be Suzuki coupled with a monocyclic heteroaryl,fused bicyclic heterocycle or heteroaryl boronate reagent followingGeneral Procedure A.

EXAMPLES

In order to illustrate the invention, the following examples areincluded. However, it is to be understood that these examples do notlimit the invention and are only meant to suggest a method of practicingthe invention. Persons skilled in the art will recognize that thechemical reactions described may be readily adapted to prepare a numberof other PI3K inhibitors of the invention, and alternative methods forpreparing the compounds of this invention are deemed to be within thescope of this invention. For example, the synthesis of non-exemplifiedcompounds according to the invention may be successfully performed bymodifications apparent to those skilled in the art, e.g., byappropriately protecting interfering groups, by utilizing other suitablereagents known in the art other than those described, and/or by makingroutine modifications of reaction conditions. Alternatively, otherreactions disclosed herein or known in the art will be recognized ashaving applicability for preparing other compounds of the invention.

Example 1 2,4-Dichloro-thieno[3,2-d]pyrimidine 3

A mixture of methyl 3-amino-2-thiophenecarboxylate 1 (13.48 g, 85.85mmol) and urea (29.75 g, 5 eq.) was heated at 190° C. for 2 hours. Thehot reaction mixture was poured onto sodium hydroxide solution and anyinsoluble material was removed by filtration. The mixture was thenacidified (HCl, 2N) to yield 1H-thieno[3,2-d]pyrimidine-2,4-dione 2 as awhite precipitate, which was collected by filtration and air dried (9.49g, 66%). ¹H NMR 400 MHz, d₆-DMSO) 6.90 (1H, d, J=5.2 Hz), 8.10 (1H, d,J=5.2 Hz), 11.60-11.10 (2H, br s).

A mixture of 1H-thieno[3,2-d]pyrimidine-2,4-dione 2 (9.49 g, 56.49 mmol)and phosphorous oxychloride (150 mL) was heated at reflux for 6 h. Thereaction mixture was then cooled and poured onto ice/water with vigorousstirring yielding a precipitate. The mixture was then filtered to yield2,4-dichloro-thieno[3,2-d]pyrimidine 3 as a white solid (8.68 g, 75%).¹H NMR (400 MHz, CDCl₃) 7.56 (1H, d, J=5.5 Hz), 8.13 (1H, d, J=5.5 Hz).

Example 2 2-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine 4

A mixture of 2,4-dichloro-thieno[3,2-d]pyrimidine 3, (8.68 g, 42.34mmol), morpholine (8.11 mL, 2.2 eq.) and MeOH (150 mL) was stirred atroom temperature for 1 h. The reaction mixture was then filtered, washedwith water and MeOH, to yield2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine 4 as a white solid(11.04 g, 100%). ¹H NMR (400 MHz, d₆-DMSO) 3.74 (4H, t, J=4.9 Hz), 3.90(4H, t, J=4.9 Hz), 7.40 (1H, d, J=5.6 Hz), 8.30 (1H, d, J=5.6 Hz).

Example 32-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10

To a suspension of 2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine 4(1.75 g, 6.85 mmol) in dry THF (40 mL) at −78° C. was added a 2.5Msolution of n-butyllithium (nBuLi) in hexane (3.3 mL, 1.2 eq.). Afterstirring for 1 h, dry DMF (796 μL, 1.5 eq.) was added. The reactionmixture was stirred for 1 h at −78° C. and then warmed slowly to roomtemperature. After a further 2 h at room temperature the reactionmixture poured onto ice/water yielding a yellow precipitate. This wascollected by filtration and air-dried to yield2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10(1.50 g, 77%). ¹H NMR (400 MHz, d₆-DMSO) 3.76 (4H, t, J=4.9), 3.95 (4H,t, J=4.9), 8.28 (1H, s), 10.20 (1H, s).

Example 3a 2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde33

To a suspension of 2-chloro-4-morpholinothieno[2,3-d]pyrimidine 38 (1.75g, 6.85 mmol) in dry THF (40 mL) at −78° C. was added a 2.5M solution ofn-butyllithium (nBuLi) in hexane (3.3 mL, 1.2 eq.). After stirring for 1h, dry DMF (796 μL, 1.5 eq.) was added. The reaction mixture was stirredfor 1 h at −78° C. and then warmed slowly to room temperature. After afurther 2 h at room temperature the reaction mixture was poured ontoice/water yielding a yellow precipitate which was collected byfiltration and air-dried to yield2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde 33 (1.50 g)MS (Q1) 284 (M+).

Example 3b 2-Chloro-6-iodo-7-methyl-4-morpholinothieno[3,2-d]pyrimidine41

To a solution of 2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidine39 (3.0 g, 11.1 mmol; prepared according to the procedure for thesynthesis of 2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine butcommencing with 3-amino-4-methyl-thiophene-2-carboxylic acid ethylester) in THF (60 mL) at −78° C. was added n-BuLi (8.9 mL, 2.5 M inEt₂O). The resulting slurry was warmed to −40° C. and stirred 50 min.The reaction mixture was then cooled to −78° C. and a solution of I₂(5.6 g, 22.2 mmol) in THF (30 mL) was added. The solution was warmed toroom temperature and stirred 5 h. The reaction was quenched by theaddition of water. The organic layer was separated and the aqueous layerwas extracted with CH₂Cl₂. The combined organics were washed withsaturated aqueous Na₂S₂O₃, dried over Na₂SO₄, filtered, and concentratedin vacuo to provide2-chloro-6-iodo-7-methyl-4-morpholinothieno[3,2-d]pyrimidine 41 (3.8 g,84% yield).

Example 3c4-(2-Chloro-6-(piperazin-1-ylmethyl)thieno[3,2-d]pyrimidin-4-yl)morpholine30

A mixture of2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10 (3.5g), 1-BOC-piperazine (2.76 g) and trimethylorthoformate (4.05 mL) wasstirred in 1,2-dichloroethane (300 mL) for 1 hr at room temperature. Tothis was added sodium triacetoxyborohydride (3.92 g) and the reactionmixture was stirred for 24 hours at room temperature. The mixture wasthen quenched with brine, extracted with dichloromethane, dried (MgSO₄)and the solvent removed in vacuo. The residue was purified using flashchromatography to yield4-(2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-piperazine-1-carboxylicacid tert-butyl ester (3.4 g). Treatment with HCl indichloromethane/methanol yielded4-(2-chloro-6-(piperazin-1-ylmethyl)thieno[3,2-d]pyrimidin-4-yl)morpholine30.

Example 3d(2-chloro-4-morpholinothieno[2,3-d]pyrimidin-6-yl)-N-methylmethanamine34

To 2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde 33 (2.0g) in 50 mL toluene and 50 mL THF was added 20 mL of 40% methylamine inH₂O. The reaction mixture was stirred at room temp under N₂ for 24hours. The solvents were removed in vacuo and the residue was dissolvedin 50 mL MeOH and 50 mL THF and the NaBH₄ added portion-wise. Thisreaction mixture was stirred at room temp under N₂ for 24 hours andcomplete reaction was confirmed by LCMS. The solvents were removed invacuo and the crude product purified by flash chromatography(EtOAc/EtOH) to give 1.12 g 34 (53% yield). MS (Q1) 300 (M+).

Example 3e(2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)-N-methylmethanamine35

2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde 10 (2.0 g)was dissolved in 50 mL toluene and 50 mL THF followed by the addition of20 mL of 40% methylamine in H₂O. The reaction mixture was stirred atroom temp under N₂ for 24 hours. The solvents were removed in vacuo andthe residue was dissolved in 50 mL methanol and 50 mL THF and the NaBH₄added portion-wise. This reaction mixture was stirred at room temp underN₂ for 24 hours and complete reaction was confirmed by LCMS. Thesolvents were removed in vacuo and the crude product purified by flashchromatography (EtOAc/EtOH) to give 1.12 g 35 (53% yield). MS (Q1) 300(M+).

Example 3f(2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)-N-methylmethanamine37

2-Chloro-7-methyl-4-morpholinothieno-[3,2-d]pyrimidine-6-carbaldehyde 36was dissolved in 20 mL toluene and 20 mL THF followed by the addition of15 mL 40% methylamine in H₂O and the reaction was stirred for 24 hours.The reaction mixture was concentrated in vacuo and the residue dissolvedin 30 mL methanol and 30 mL THF followed by the addition of NaBH₄. Thereaction was stirred at room temp for at least 24 hours and productformation was confirmed by LCMS. The solvents were removed in vacuo andthe crude product purified by flash chromatography to give 2.53 g(2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)-N-methylmethanamine37 (70% yield) MS (Q1) 314 (M)+

Example 44-(2-Chloro-6-((4-(methylsulfonyl)piperazin-1-ypmethyl)thieno[3,2-d]pyrimidin-4-yl)morpholine31

Reaction between N-BOC-piperazine and methane sulfonyl chloride indichloromethane and triethylamine yielded4-methanesulfonyl-piperazine-1-carboxylic acid tert-butyl ester.Cleavage of the BOC protecting group using HCl (2M) in dichloromethaneyielded 1-methanesulfonyl-piperazine HCl salt.

A mixture of2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10(1.00 g), 1-methanesulfonyl-piperazine (750 mg) andtrimethylorthoformate (3.80 mL) was stirred in 1,2-dichloroethane (30mL) for 6 hrs at room temperature. To this was added sodiumtriacetoxyborohydride (900 mg) and the reaction mixture was stirred for24 hours at room temperature. The mixture was then quenched with brine,extracted with dichloromethane, dried (MgSO₄) and the solvent removed invacuo. The residue was triturated with hot ethyl acetate to yield4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine31 as a white solid (1.01 g).

Example 52-Chloro-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[2,3-d]pyrimidine32

Reaction between 1-methanesulfonyl-piperazine HCL salt and2-chloro-4-morpholin-4-yl-thieno[2,3-d]pyrimidine-6-carbaldehyde 33using General Procedure C Yielded2-chloro-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[2,3-d]pyrimidine.

Example 6 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole7-route 1

Intermediate 7 was prepared according to the methods of US 2008/0076768;US 2008/0076758; WO 2006/046031, incorporated by reference herein.

Example 81-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole40

Intermediate 40 was prepared according to the methods of US2008/0039459; US 2008/0076768; US 2008/0076758; WO 2006/046031,incorporated by reference herein.

Example 102-(1H-Indazol-4-yl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde11

A mixture of2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10 (100mg, 0.35 mmol),4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole (70) (95mg, 0.39 mmol) and sodium carbonate (112 mg) were suspended in toluene(2.5 mL), ethanol (1.5 mL) and water (0.7 mL). To this was addedbis(triphenylphosphine)palladium(II) chloride (13.5 mg) and the reactionvessel was flushed with argon. The reaction mixture was microwaved at120° C. for 1 h and then partitioned between DCM and water, the organiclayer was washed with brine, dried over magnesium sulfate, filtered andevaporated in vacuo. The resulting residue was purified using flashchromatography to yield2-(1H-indazol-4-yl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde11 (97 mg).

Example 114-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine(Formula Ia, GDC-0941):

A mixture of4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine31 from Example 4 (2.00 g),4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole 7 (2.26 g),toluene (24 mL), ethanol (12 mL), water(6 mL), sodium carbonate (1.72 g)and PdCl₂(PPh₃)₂ (325 mg) was heated to 130° C. in the microwave for 90minutes (US 2008/0076768; WO 2006/046031, incorporated by referenceherein).

The reaction mixture was cooled, diluted with chloroform, washed withbrine, dried (MgSO₄) and the solvent removed in vacuo. The residue waspurified using flash chromatography (ethyl acetate then 5% ethylacetate/methanol) and then trituration with ether yielded Formula Iacompound, GDC-0941 (1.4 g). MS data: (ESI+): MH+ 514. NMR data: (CDCl3):2.67-2.71 (4H, m), 2.81 (3H, s), 3.29-3.33 (4H, m), 3.89 (2H, s),3.89-3.93 (4H, m), 4.08-4.12 (4H, m),7.41 (1H, s), 7.51 (1H, t, J=7.2),7.60 (1H, d, J=8.3), 8.28 (1H, d, J=7.5), 9.02 (1H, s), 10.10 (1H, br)

Example 124-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine(Formula IIa):

2-Chloro-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[2,3-d]pyrimidine32 from Example 5 was reacted with4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole 7 andGeneral Procedure A to give Formula IIa compound which was purifiedusing flash chromatography (US 2008/0076758; WO 2006/046031,incorporated by reference herein). 400 MHz 1H NMR CDCl3: 2.67 (m, 4H,2×CH2), 2.81 (s, 3H, CH3), 3.30 (m, 4H, 2×CH2), 3.83 (s, 2H, CH2),3.92-3.94 (m, 4H, 2×CH2), 3.98-4.00 (m, 4H, 2×CH2), 7.17 (s, H, ArH),7.50 (t, H, ArH, J=7.81 Hz), 7.59 (d, H, ArH, J=8.31 Hz), 8.31 (d, H,ArH, J=6.98 Hz), 10.12 (sbr, H, NH). MH+=514.10

Example 12a(S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one(Formula Ib):

2-Chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde 36(495 mg) was reacted with Boc-piperazine via General Procedure B-3 togive tert-butyl4-((2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazine-1-carboxylate.

Tert-butyl44(2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazine-1-carboxylate(777 mg) was subjected to General Procedure E to give the HCl salt of2-chloro-7-methyl-4-morpholino-6-((piperazin-1-yl)methyl)thieno[3,2-d]pyrimidine.The HCl salt of2-chloro-7-methyl-4-morpholino-6-((piperazin-1-yl)methyl)thieno[3,2-d]pyrimidine(590 mg) was reacted with lactic acid via General Procedure B-2 to give(S)-1-(4-((2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one.

(S)-1-(4-((2-Chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one(60 mg) was reacted with 50 mg of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine viaGeneral Procedure A-2 to give 10 mg of Formula Ib (WO 2008/070740,incorporated by reference). MS (Q1) 499.3 (M)+.

Example 13 p110α (alpha) PI3K Binding Assay

Binding Assays: Initial polarization experiments were performed on anAnalyst HT 96-384 (Molecular Devices Corp, Sunnyvale, Calif.). Samplesfor fluorescence polarization affinity measurements were prepared byaddition of 1:3 serial dilutions of p110alpha PI3K (Upstate CellSignaling Solutions, Charlottesville, Va.) starting at a finalconcentration of 20 ug/mL in polarization buffer (10 mM Tris pH 7.5, 50mM NaCl, 4 mM MgCl₂, 0.05% Chaps, and 1 mM DTT) to 10 mM PIP₂(Echelon-Inc., Salt Lake City, Utah) final concentration. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah) 100 nM and 5 nM final concentrations respectively.Read with standard cut-off filters for the rhodamine fluorophore(λex=530 nm; λem=590 nm) in 384-well black low volume Proxiplates(PerkinElmer, Wellesley, Mass.) Fluorescence polarization values wereplotted as a function of the protein concentration, and the EC₅₀ valueswere obtained by fitting the data to a 4-parameter equation usingKaleidaGraph software (Synergy software, Reading, Pa.). This experimentalso establishes the appropriate protein concentration to use insubsequent competition experiments with inhibitors.

Inhibitor IC₅₀ values were determined by addition of the 0.04 mg/mLp110alpha PI3K (final concentration) combined with PIP₂ (10 mM finalconcentration) to wells containing 1:3 serial dilutions of theantagonists in a final concentration of 25 mM ATP (Cell SignalingTechnology, Inc., Danvers, Mass.) in the polarization buffer. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah) 100 nM and 5 nM final concentrations respectively.Read with standard cut-off filters for the rhodamine fluorophore(λex=530 nm; λem=590 nm) in 384-well black low volume proxi plates(PerkinElmer, Wellesley, Mass.) Fluorescence polarization values wereplotted as a function of the antagonist concentration, and the IC₅₀values were obtained by fitting the data to a 4-parameter equation inAssay Explorer software (MDL, San Ramon, Calif.).

Alternatively, inhibition of PI3K was determined in a radiometric assayusing purified, recombinant enzyme and ATP at a concentration of 1 uM.The compound was serially diluted in 100% DMSO. The kinase reaction wasincubated for 1 h at room temperature, and the reaction was terminatedby the addition of PBS. IC₅₀ values were subsequently determined usingsigmoidal dose-response curve fit (variable slope).

Example 14 In Vitro Cell Proliferation Assay

Efficacy of Formula I or II compounds were measured by a cellproliferation assay employing the following protocol (Promega Corp.Technical Bulletin TB288; Mendoza et al (2002) Cancer Res.62:5485-5488). The Cell-Titer Glo assay reagents and protocol arecommercially available (Promega). The assay assesses the ability ofcompounds to get into cells and inhibit cell proliferation. The assayprinciple is the determination of the number of viable cells present byquantitating the ATP present. Cell-Titer Glo is the reagent used forthis quantitation. It is a homogenous assay where addition of theCell-Titer Glo results in cell lysis and generation of a luminescentsignal through the luciferase reaction. The luminescent signal isproportional to the amount of ATP present.

Cells: see FIGS. 1A-C for cell lines and tumor type

DMSO and Media Plates: 96-well conical bottom polypropylene plates fromNunc (cat. #249946)

Cell Plates: 384-well black, clear bottom (microclear), TC plates withlid from Falcon (353962)

Cell Culture Medium: RPMI or DMEM high glucose, 10% Fetal Bovine Serum,2 mM L-Glutamine, P/S

Cell Titer-Glo: Promega (cat. #G7572)

Procedure:

Day 1—Seed Cell Plates, Harvest cells, Seed PC3 cells at 1000 cells per54 μl per well into 384 well Cell Plates for 3 days assay. Incubate O/Nat 37 C, 5% CO2.

Day 2—Add Drug to Cells, Compound Dilution, DMSO Plates (serial 1:2 for9 points), Add 20 ul compounds at 10 mM in the 2nd column of 96 wellplate. Perform serial 1:2 across the plate (10 μl+10 μl 100% DMSO) for atotal of 9 points using Precision. Media Plates (1:50 dilution) Add 147μl of Media into all wells. Transfer 3 μl of DMSO+compound from eachwell in the DMSO Plate to each corresponding well on Media Plate usingRapidplate. For 2 drug combo studies, transfer one drug1.5 μl ofDMSO+compound from each well in the DMSO Plate to each correspondingwell on Media Plate using Rapidplate. Then, transfer another drug 1.5 ulto the medium plate.

Drug Addition to Cells, Cell Plate (1:10 dilution), Add 6 μl ofmedia+compound directly to cells (54 μl of media on the cells already).Incubate 3 days at 37 C, 5% CO2 in an incubator that will not be openedoften.

Day 5—Develop Plates, Thaw Cell Titer Glo Buffer at room temperature.Remove Cell Plates from 37° C. and equilibrate to room temperature. forabout 30 minutes. Add Cell Titer Glo Buffer to Cell Titer Glo Substrate(bottle to bottle). Add 30 μl Cell Titer Glo Reagent to each well ofcells. Place on plate shaker for about 30 minutes. Read luminescence onAnalyst HT Plate Reader (half second per well).

Cell viability assays and combination assays: Cells were seeded at1000-2000 cells/well in 384-well plates for 16 h. On day two, nineserial 1:2 compound dilutions were made in DMSO in a 96 well plate. Thecompounds were further diluted into growth media using a Rapidplaterobot (Zymark Corp., Hopkinton, Mass.). The diluted compounds were thenadded to quadruplicate wells in 384-well cell plates and incubated at 37C and 5% CO2. After 4 days, relative numbers of viable cells weremeasured by luminescence using Cell-Titer Glo (Promega) according to themanufacturer's instructions and read on a Wallac Multilabel Reader(PerkinElmer, Foster City). EC50 values were calculated using Prism 4.0software (GraphPad, San Diego). Drugs in combination assays were dosedstarting at 4× EC50 concentrations. If cases where the EC50 of the drugwas >2.5 μM, the highest concentration used was 10 μM. PI3K inhibitorsand chemotherapeutic agents were added simultaneously or separated by 4hours (one before the other) in all assays.

An additional exemplary in vitro cell proliferation assay includes thefollowing steps:

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells (seeFIGS. 1A-C for cell lines and tumor type) in medium was deposited ineach well of a 384-well, opaque-walled plate.

2. Control wells were prepared containing medium and without cells.

3. The compound was added to the experimental wells and incubated for3-5 days.

4. The plates were equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well was added.

6. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence was recorded and reported in graphs as RLU=relativeluminescence units.

Alternatively, cells were seeded at optimal density in a 96 well plateand incubated for 4 days in the presence of test compound. Alamar Blue™was subsequently added to the assay medium, and cells were incubated for6 h before reading at 544 nm excitation, 590 nm emission. EC₅₀ valueswere calculated using a sigmoidal dose response curve fit.

Example 15 FACS Annexin V/PI Assay

Cells (2×10⁶) were placed in a 10 cm tissue culture plate. After 16hours, the cells were exposed to 0.1% DMSO (control) or Formula Ia(containing 0.1% DMSO) for 48 hours. Cells were then removed from theplate using trypsin and washed once with PBS. To detect apoptosis, cells(MB361, PC3) were resuspended in binding buffer (10 mM Hepes/NaOH [pH7.4], 140 mM NaCl, and 2.5 mM CaCl₂) at 1×10⁶ cells/mL and immediatelystained with 5 μL annexin V-FITC (BD Pharmingen; Franklin Lakes, N.J.)and 500 μL propidium iodide (PI) solution containing 50 μg/mL PI(Sigma), 0.2 mg/mL RNase solution (Sigma), and 0.1% Triton-X (Sigma) inPBS. The mixture was incubated at room temperature for 30 minutes andcells were analyzed with a flow cytometer (BD Biosciences; San Jose,Calif.).

Example 16 Acinar Morphogenesis in 3D Culture of HER2+BT474M1 Cells

The biological activity of PI3K inhibitors and the most effectivetherapeutic combinations of PI3K and HER family inhibitors inHER2-amplified breast cancer cells were determined in 3D cell cultureperformed using the overlay method. Formula Ia compound was used as asuspension in dimethylsulfoxide at a concentration of 50 mM. Heregulinbeta-1₁₇₇₋₂₄₄ (hereafter referred to as HRG) was provided at a storageconcentration of 225.8 μM. BT474M1 cells were treated with 20 μg/mltrastuzumab, 25 μg/m1pertuzumab, 250 nM Formula Ia compound, or 250 nMFormula IIa compound in 3D culture. Cell viability was determined bymeasuring cellular ATP levels using the Cell Titer-Glo Luminescent CellViability Assay (Promega). Readings were based on the average of 3replicates per assay condition. Morphogenesis was quantified bycalculating the extent of bud-formation from phase-contrast images. 100Acini in each assay condition, typically 9 days in duration, were scoredfor the number of buds formed on the acinar surface. Culture media isrefreshed every 3 days.

In a titration curve, BT474M1 cells were treated with incremental dosesof Formula Ia to determine an optimal concentration that effectivelyinhibited markers downstream of PI3K (AKT) and led to an overalldecrease in cell viability (Cell Titer-Glo). BT474M1 cells were derivedfrom a BT474 parental cell line purchased through the American TypeCulture Collection. Cells were passaged through mice to obtain a viableestrogen-dependent cell line suitable for in vitro and in vivo studies.

All 3D assays were performed using the “overlay method” as described(Lee et al (2007) Nat Methods. 4:359-65). Forty-eight-well dishes werecoated evenly with 100 ul of growth factor-reduced Matrigel (BDBiosciences) on ice. Plates were subsequently transferred to a 37° C.incubator for 20 minutes to allow for matrix polymerization. BT474M1cells were harvested and 10,000 cells/well were seeded ontoMatrigel-coated dishes. Cells were cultivated in growth mediumsupplemented with 5% Matrigel and corresponding drugs or ligand. Assayswere typically 9-10 days in duration, and growth medium was replacedevery 3 days. Phase-contrast images were recorded with a Sony DigitalCamera (DXC-5500) adapted to a Leica DMIL microscope. Cell viability wasdetermined by measuring cellular ATP levels using the Cell Titer-GloLuminescent Cell Viability Assay (Promega). Readings were assessed by aluminometer and based on the average of 3 replicates per assaycondition. Morphogenesis was quantified by calculating the extent ofbud-formation from phase-contrast images. One hundred acini in eachassay condition were scored for the number of buds formed on the acinarsurface. Scores were totaled and grouped into categories of: 0-1, 2-3,or ≧4 buds per acini.

TABLE 1 PI3K inhibitor combinations evaluated in 3D cell cultureCombinations Target(s) 1  20 μg/ml trastuzumab Her2 (extracellularsubdomain IV) 250 nM Formula Ia PI3K 2  25 μg/ml pertuzumab Her2(extracellular subdomain II) 250 nM Formula Ia PI3K 3  20 μg/mltrastuzumab Her2 (extracellular subdomain II)  25 μg/ml pertuzumab Her2(extracellular subdomain IV) 250 nM Formula Ia PI3K

Example 17 In Vivo Tumor Xenograft

Animals suitable for transgenic experiments can be obtained fromstandard commercial sources. Groups of female CD-1 nude mice (CharlesRiver Laboratory) were implanted subcutaneously in the hind flank with20 million MDA-MB-361.1 (PI3K mutant) breast cancer cells with matrigeland 0.36 mg of estrogen implants per mouse. Groups of female NMRI nu/numice (Janvier) were implanted with 150 mm3 fragments of MAXF 401(Her2+/ER+/PR+) or MAXF 1162 (Her2+/ER+/PR+) primary breast tumors(biopsied directly from two individual breast cancer patients) withmatrigel and 0.36 mg of estrogen pellets per mouse. Groups of femaleHRLN nu/nu (Harlan Labs) were implanted with 10 million MCF-7 (PI3Kmutant) breast cancer cells with matrigel and 0.36 mg of estrogenpellets per mouse. Groups of female athymic nu/nu mice (Charles RiverLaboratory) were implanted with 15 million NCI-H2122 (K-Ras mutant)non-small cell lung cancer cells and matrigel per mouse. Mousexenografts were dosed at day 1 with drug, drug combination, or vehicleaccording to the schedule specified for each tumor model. Docetaxel wasadministered intravenously, B20-1.4 was administered intraperitonealyand Formula Ia and IIa were delivered per os by oral gavage. Tumor sizeswere recorded twice weekly over the course of the study. Mouse bodyweights were also recorded twice weekly, and the mice were observedregularly. Tumor volume was measured in two dimensions (length andwidth) using Ultra Cal-IV calipers (Model 54-10-111; Fred V. Fowler Co.,Inc.; Newton, Mass.) and analyzed using Excel v.11.2 (MicrosoftCorporation; Redmond, Wash.). Tumor inhibition graphs were plotted usingKaleidaGraph, Version 3.6 (Synergy Software; Reading, Pa.). The tumorvolume was calculated with formula: Tumor size (mm³)=(longermeasurement×shorter measurement²)×0.5

Animal body weights were measured using an Adventurera Pro AV812 scale(Ohaus Corporation; Pine Brook, N.J.). Graphs were generated usingKaleidaGraph Version 3.6. Percent weight change was calculated usingformula: Group percent weight change=(1−(initial weight/newweight))×100.

Mice whose tumor volume exceeded 2000 mm³ or whose body weight losswas >20% of their starting weight were promptly euthanized according toregulatory guidance.

The percent tumor growth inhibition (% INH) at the end of study (EOS)was calculated using formula: % INH=100∴(EOS mean volume of tumors inanimals given vehicle−EOS mean volume of tumors in animals given thedrug)/EOS mean volume of tumors in animals given vehicle.

Tumor incidence (TI) was determined based on the number of measurabletumors remaining in each group at the end of the study. A partialresponse (PR) was defined as a >50% but <100% reduction in tumor volume,compared with the starting tumor volume, observed on any day of thestudy. A complete response (CR) was defined as a 100% reduction in tumorvolume, compared with the initial tumor volume, observed on any day ofthe study. Data were analyzed and p-values were determined using theDunnett's test with JMP statistical software, version 5.1.2 (SASInstitute; Cary, N.C.). Individual tumor volumes at end of study andmean tumor volume ±SEM values were calculated using JMP statisticalsoftware, version 5.1.2. Body weight data were graphed based on the meanpercentage of change from initial body weights ±SEM.

Example 18 Phospho AKT Induction Assay

In a 6-well tissue culture plate cells were seeded at 5×10⁵ cells perwell overnight. Cells were treated with an EC₈₀ of the chemotherapeuticagent. Following treatment, cells were washed once with cold PBS andlysed in 1× Cell Extraction Buffer from Biosource (Carlsbad, Calif.)supplemented with protease inhibitors (Roche, Mannheim, Germany), 1 mMPMSF, and Phosphatase Inhibitor Cocktails 1 and 2 from Sigma (St. Louis,Mo.). Determination of protein concentration was performed using thePierce BCA Protein Assay Kit (Rockford, Ill.). Levels of pAkt (Ser⁴⁷³)and total Akt were assessed using bead kits from Biosource (Carlsbad,Calif.) and the Luminex Bio-Plex system (Bio-Rad, Hercules, Calif.).

The foregoing description is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will be readily apparent to those skilled in the art, it is notdesired to limit the invention to the exact construction and processshown as described above. Accordingly, all suitable modifications andequivalents may be considered to fall within the scope of the inventionas defined by the claims that follow.

We claim:
 1. A method for determining compounds to be used in combination for the treatment of cancer comprising: a) administering a therapeutic combination of a compound having Formula I or II, and a chemotherapeutic agent to HER2-amplified breast cancer cells in laminin-rich, reconstituted basement membrane media, wherein the chemotherapeutic agent targets, binds to, or modulates a HER2 receptor, and b) measuring inhibition of cellular proliferation wherein nonmalignant and malignant mammary cells are discriminated by one or more phenotypic difference selected from cell viability and acinar morphogenesis; wherein the Formula I or II compound is selected from a compound of Formula Ia, Formula Ib and Formula IIa:

or stereoisomers, geometric isomers, tautomers, or pharmaceutically acceptable salts thereof.
 2. The method of claim 1 wherein the Formula I or II compound is a compound of Formula Ia.
 3. The method of claim 1 wherein the chemotherapeutic agent is an anti-HER2 antibody.
 4. The method of claim 3 wherein the anti-HER2 antibody is selected from trastuzumab and pertuzumab.
 5. The method of claim 1 wherein the chemotherapeutic agent is selected from erlotinib, docetaxel, 5-FU, gemcitabine, PD-0325901, cisplatin, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, temozolomide, tamoxifen, doxorubicin, Akti-½ , HPPD, rapamycin, and lapatinib.
 6. The method of claim 1 wherein the laminin-rich, reconstituted basement membrane media is Engelbreth-Holm-Swarm extracellular matrix extract.
 7. The method of claim 1 wherein the Formula I or II compound is a compound of Formula Ib.
 8. The method of claim 1 wherein the Formula I or II compound is a compound of Formula IIa.
 9. The method of claim 1, wherein the therapeutic combination comprises a compound having Formula I or II, a first chemotherapeutic agent and second chemotherapeutic agent.
 10. The method of claim 2, wherein the chemotherapeutic agent is trastuzumab.
 11. The method of claim 2, wherein the chemotherapeutic agent is pertuzumab.
 12. The method of claim 2, wherein the chemotherapeutic agent is lapatinib.
 13. The method of claim 7, wherein the chemotherapeutic agent is trastuzumab.
 14. The method of claim 7, wherein the chemotherapeutic agent is pertuzumab.
 15. The method of claim 7, wherein the chemotherapeutic agent is lapatinib. 